Pyrazolopyridazines and methods for treating retinal-degenerative diseases and hearing loss associated with usher syndrome

ABSTRACT

Compounds, compositions and methods for the treatment of retinal degenerative diseases, such as retinitis pigmentosa, Leber&#39;s congenital Amaurosis, Syndromic retinal degenerations, age-related macular degeneration and Usher Syndrome, and hearing loss associated with Usher Syndrome are described herein.

This application is a continuation of U.S. application Ser. No.14/730,994, filed Jun. 4, 2015, now U.S. Pat. No. 9,260,381, which is adivision of U.S. application Ser. No. 13/791,205, filed Mar. 8, 2013,now U.S. Pat. No. 9,079,909, which is a continuation of InternationalPatent Application No. PCT/US2012/34959, filed on Apr. 25, 2012, whichclaims the benefit of U.S. Provisional Application No. 61/478,642, filedApr. 25, 2011, U.S. Provisional Application No. 61/537,908, filed Sep.22, 2011, and U.S. Provisional Application No. 61/576,471, filed Dec.16, 2011, the disclosure of each of which is hereby incorporated byreference herein in its entirety.

BACKGROUND OF THE INVENTION

Usher Syndrome, a rare genetic disorder and a leading cause of deafnessand blindness, is associated with a mutation in any one of ten genes.Other names for the syndrome include Hallgren Syndrome, Usher-HallgrenSyndrome, RP-Dysacusis Syndrome, and Dystrophia Retinae DysacusisSyndrome.

Usher Syndrome is characterized by deafness and gradual vision loss. Thehearing loss is associated with inner ear defects, whereas the visionloss is associated with retinitis pigmentosa (RP), a degeneration of theretinal cells. Usually, the rod cells of the retina are affected first,leading to early night blindness and the gradual loss of peripheralvision. Some cases involve early degeneration of the cone cells of themacula, leading to a loss of central acuity. In some cases, thesufferer's foveal vision is spared, leading to “doughnut vision,” inwhich central and peripheral vision remain intact, but interrupted by aring of blindness.

Usher Syndrome has three clinical subtypes, denoted: I, II and III.Usher I subjects are born profoundly deaf, begin to lose vision withinten years and exhibit balance difficulties. They are slow to learn towalk as children, due to vestibular abnormalities. Usher II subjectssuffer lesser hearing loss, do not suffer physical imbalance and beginto lose vision in adolescence. Much of their hearing can be preservedinto middle age. Usher III subjects suffer gradual loss of hearing andvision and can suffer physical imbalance.

Usher Syndrome is a variable condition; the degree of severity is nottightly linked to subtype. For example, an Usher III subject might beasymptomatic in childhood, but develop profound hearing and vision lossby early to mid adulthood. Substantial visual impairment prior to age 50is common in Usher III subjects. An Usher I subject, on the other hand,might be deaf from birth, but sustain good central vision into old age.

SUMMARY OF THE INVENTION

The invention provides compounds of Formula I:

and pharmaceutically acceptable salts thereof,

-   -   wherein R is fluoro, chloro, iodo, methyl, methoxy, cyano,        trifluoromethyl, or —(CO)NH(CH₃).

The invention also provides compounds of Formula II:

and pharmaceutically acceptable salts thereof,

-   -   wherein Hal is —Cl, —F, —I, or —Br;    -   x is an integer ranging from 0 to 5;    -   each R₁ is independently —Cl, —F, —I, —Br, —C₁-C₃ alkyl,        —O—C₁-C₃ alkyl, —CN, —CF₃, —C(O)NH(CH₃), or —C≡CCH₂OH;    -   y is an integer ranging from 0 to 5;    -   each R₂ is independently —Cl, —F, —Br, —C₁-C₃ alkyl, —O—C₁-C₃        alkyl, —CN, —CF₃, —C(O)NH(CH₃), or —C≡CCH₂OH;    -   R₃ is —H, —C₁-C₆ alkyl, —(C₁-C₆ alkylene)-OH, —(C₁-C₆        alkylene)-phenyl, —(C₁-C₆ alkylene)-O—(C₁-C₆ alkyl), —C₂-C₆        alkenyl, —(C₁-C₆ alkylene)-C(O)R₄, —(C₁-C₆ alkylene)-R₅,

-   -   R₄ is —OH, —O—(C₁-C₆ alkyl), —NH₂, —NH(C₁-C₆ alkyl), —NH((C₁-C₆        alkylene)-OH), —NH((C₁-C₆ alkylene)N(C₁-C₆ alkyl)₂), —N(C₁-C₆        alkyl)((C₁-C₆ alkylene)-CN), —N(C₁-C₆ alkyl)((C₁-C₆        alkylene)N(C₁-C₆ alkyl)₂), —NH(C₁-C₆ alkylene)-O—(C₁-C₆ alkyl),

-   -   a is an integer ranging from 0 to 10;    -   b is an integer ranging from 0 to 8;    -   c is an integer ranging from 0 to 6; and    -   R₅ is

The invention additionally provides compounds of Formula III:

and pharmaceutically acceptable salts thereof,

-   -   wherein Hal is —Cl, —F, —I, or —Br;    -   x is an integer ranging from 0 to 5;    -   each R₁ is independently —Cl, —F, —I, —Br, —C₁-C₃ alkyl,        —O—C₁-C₃ alkyl, —CN, —CF₃, —C(O)NH(CH₃), or —C≡CCH₂OH;    -   R₃ is —H, —C₁-C₆ alkyl, —(C₁-C₆ alkylene)-OH, —(C₁-C₆        alkylene)-phenyl, —(C₁-C₆ alkylene)-O—(C₁-C₆ alkyl), —C₂-C₆        alkenyl, —(C₁-C₆ alkylene)-C(O)R₄, —(C₁-C₆ alkylene)-R₅,

-   -   R₄ is —OH, —O—(C₁-C₆ alkyl), —NH₂, —NH(C₁-C₆ alkyl), —NH((C₁-C₆        alkylene)-OH), —NH((C₁-C₆ alkylene)N(C₁-C₆ alkyl)₂), —N(C₁-C₆        alkyl)((C₁-C₆ alkylene)-CN), —N(C₁-C₆ alkyl)((C₁-C₆        alkylene)N(C₁-C₆ alkyl)₂), —NH(C₁-C₆ alkylene)-O—(C₁-C₆ alkyl),

-   -   a is an integer ranging from 0 to 10;    -   b is an integer ranging from 0 to 8;    -   c is an integer ranging from 0 to 6;    -   R₅ is

-   -   wherein each R₆ and R₇ is independently —H or —I, wherein at        least one of R₆ and R₇ is —I,    -   and wherein when R₃ is —C₁-C₃ alkyl, R₇ is —H.

The invention further provides compounds having the followingstructures:

and pharmaceutically acceptable salts thereof.

Compounds 1-35, 37-39, 42, 45, and 47-97 are illustrative compounds ofFormula II.

Each of the compounds of Formulas I, II, and III and above compounds1-35, 37-39, 42, 45, and 47-97, or a pharmaceutically acceptable saltthereof, (a “Pyrazolopyridazine compound”) is useful for treating aretinal degenerative disease or hearing loss associated with UsherSyndrome.

The invention further provides compositions comprising an effectiveamount of a Pyrazolopyridazine compound and a pharmaceuticallyacceptable carrier or vehicle. The compositions are useful for treatinga retinal degenerative disease or hearing loss associated with UsherSyndrome.

The invention further provides methods for treating a retinaldegenerative disease, comprising administering to a subject in needthereof an effective amount of a Pyrazolopyridazine compound.

The invention still further provides methods for treating hearing lossassociated with Usher Syndrome, comprising administering to a subject inneed thereof an effective amount of a Pyrazolopyridazine compound.

The invention still further provides compounds of Formula IV:

and salts thereof,

wherein R₈ is —C₁-C₃ alkyl.

The invention still further provides Compound 43, an illustrativecompound of Formula IV, which has the structure:

and salts thereof.

Compounds of Formula IV and salts thereof are useful as intermediatesfor synthesizing Pyrazolopyridazine compounds.

The invention still further provides Compound 44, which has thestructure:

and salts thereof.

Compound 44 is useful as a probe for identifying proteins that bind toits bisphenyl pyrazolopyridazine moiety.

The invention still further provides Compound 46, which has thestructure:

and pharmaceutically acceptable salts thereof.

Compound 46 or a pharmaceutically acceptable salt thereof is also usefulfor treating a retinal degenerative disease or hearing loss associatedwith Usher Syndrome.

The invention further provides compositions comprising an effectiveamount of Compound 46, or a pharmaceutically acceptable salt thereof,and a pharmaceutically acceptable carrier or vehicle. The compositionsare useful for treating a retinal degenerative disease or hearing lossassociated with Usher Syndrome.

The invention further provides methods for treating a retinaldegenerative disease, comprising administering to a subject in needthereof an effective amount of Compound 46 or a pharmaceuticallyacceptable salt thereof.

The invention still further provides methods for treating hearing lossassociated with Usher Syndrome comprising, administering to a subject inneed thereof an effective amount of Compound 46 or a pharmaceuticallyacceptable salt thereof.

Each of the following is a “compound of the invention”: aPyrazolopyridazine compound; Compound 1-35, 37-39 or 42-97, or a saltthereof; and a compound having the structure of Formula I, II, or III,or a salt thereof.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 illustrates density of N48K Clarin-1 expression in cells.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides compounds of the invention, compositionscomprising a compound of the invention, and methods for treating aretinal degenerative disease or hearing loss associated with UsherSyndrome, comprising administering a Pyrazolopyridazine compound orCompound 46 or a pharmaceutically acceptable salt thereof.

Compounds of the Invention

The term “alkyl” refers to a straight or branched saturated hydrocarbongroup. Illustrative alkyl groups include —CH₃, —CH₂CH₃, —CH₂CH₂CH₃,—CH(CH₃)₂, —CH₂CH₂CH₂CH₃, —CH(CH₃)CH₂CH₃, —CH₂CH(CH₃)₂, —C(CH₃)₃,—CH₂CH₂CH₂CH₂CH₃, —CH(CH₃)CH₂CH₂CH₃, —CH₂CH₂CH(CH₃)₂, —CH₂C(CH₃)₃,—CH₂CH₂CH₂CH₂CH₃, —CH(CH₃)CH₂CH₂CH₃, —CH₂CH₂CH(CH₃)₂ and —CH(CH₃)C(CH₃)₃groups.

The term “alkylene” refers to an alkyl group bonded to another atom orgroup. Illustrative alkylene groups include —CH₂—, —CH₂CH₂—,—CH₂CH₂CH₂—, —C(CH₃)₂—, —CH(CH₃)—, —CH₂CH₂CH₂CH₂—, —CH(CH₃)CH₂CH₂—,—CH₂C(CH₃)₂—, —C(CH₃)₂CH₂—, —CH₂CH₂CH₂CH₂CH₂—, —CH(CH₃)CH₂CH₂CH₂—,—CH₂CH₂C(CH₃)₂—, —CH₂CH(CH₃)CH₂CH₂—, —CH₂CH₂CH(CH₃)CH₂—,—CH₂CH₂CH₂CH₂CH₂CH₂—, —CH(CH₃)CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂C(CH₃)₂—,—CH₂CH(CH₃)CH₂CH₂CH₂—, —CH₂CH₂CH₂CH(CH₃)CH₂— and —C(CH₃)₂C(CH₃)₂—groups.

The term “alkenyl” refers to a straight or branched hydrocarbon grouphaving one or more double bonds. Illustrative alkenyl groups include—CH═CH₂, —CH₂CH═CH₂, cis-CH═CHCH₃, trans-CH═CHCH₃, —C(CH₃)═CH₂,cis-CH═CHCH₂CH₃, trans-CH═CHCH₂CH₃, cis-CH₂CH═CHCH₃, trans-CH₂CH═CHCH₃,—CH₂CH₂CH═CH₂, cis-CH═CHCH₂CH₂CH₃, trans-CH═CHCH₂CH₂CH₃,cis-CH₂CH₂CH═CHCH₃, trans-CH₂CH₂CH═CHCH₃, —CH₂CH₂CH₂CH═CH₂,—CH₂CH═C(CH₃)₂, cis-CH═CHCH₂CH₂CH₂CH₃, trans-CH═CHCH₂CH₂CH₂CH₃,cis-CH₂CH₂CH₂CH═CHCH₃, trans-CH₂CH₂CH₂CH═CHCH₃, —CH₂CH₂CH₂CH₂CH═CH₂, and—CH₂CH₂CH═C(CH₃)₂, groups.

The word “about” when immediately preceding a numerical value means arange of plus or minus 10% of that value, e.g., “about 100 mg” means 90mg to 110 mg, “about 300 mg” means 270 mg to 330 mg, etc.

Abbreviations

-   APCI Atmospheric Pressure Chemical Ionization-   DAPI 4′,6-diamidino-2-phenylindole-   DIPEA diisopropylethylamine-   DMEM Dulbecco's Modified Eagle Medium-   DMF dimethylformamide-   DMSO Dimethyl sulfoxide-   EDAC 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride-   ESI Electrospray ionization-   ESI-TOF Electrospray ionization-Time-of-flight-   HATU 2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium    hexafluorophosphate-   HOPO 2-hydroxypyridine-N-oxide-   HPLC High-performance liquid chromatography-   LCMS Liquid chromatography-mass spectrometry-   LDA lithium diisopropyl amide-   m/z Mass-to-charge ratio-   MALDI-TOF Matrix Assisted Laser Desorption Ionization-Time-of-flight-   MS Mass spectrometry-   PBS phosphate-buffered saline-   Rt Retention time-   SDS sodium dodecylsulfate-   THF tetrahydrofuran

Compounds of Formula I

In one embodiment, the Pyrazolopyridazine compound is a compound ofFormula I:

or a pharmaceutically acceptable salt thereof,wherein R is fluoro, chloro, iodo, methyl, methoxy, cyano,trifluoromethyl, or —(CO)NH(CH₃). In one embodiment, R of Formula I isin the para position relative to the pyrazolopyridazino ring system. Inone embodiment, R of Formula I is in the meta position relative to thepyrazolopyridazino ring system. In one embodiment, R of Formula I is inthe ortho position relative to the pyrazolopyridazino ring system.

Compounds of Formula II

The invention also provides compounds of Formula II:

and pharmaceutically acceptable salts thereof,

-   -   wherein Hal is —Cl, —F, —I, or —Br;    -   x is an integer ranging from 0 to 5;    -   each R₁ is independently —Cl, —F, —I, —Br, —C₁-C₃ alkyl,        —O—C₁-C₃ alkyl, —CN, —CF₃, —C(O)NH(CH₃), or —C≡CCH₂OH;    -   y is an integer ranging from 0 to 5;    -   each R₂ is independently —Cl, —F, —Br, —C₁-C₃ alkyl, —O—C₁-C₃        alkyl, —CN, —CF₃, —C(O)NH(CH₃), or —C≡CCH₂OH;    -   R₃ is —H, —C₁-C₆ alkyl, —(C₁-C₆ alkylene)-OH, —(C₁-C₆        alkylene)-phenyl, —(C₁-C₆ alkylene)-O—(C₁-C₆ alkyl), —C₂-C₆        alkenyl, —(C₁-C₆ alkylene)-C(O)R₄, —(C₁-C₆ alkylene)-R₅,

-   -   R₄ is —OH, —O—(C₁-C₆ alkyl), —NH₂, —NH(C₁-C₆ alkyl), —NH((C₁-C₆        alkylene)-OH), —NH((C₁-C₆ alkylene)N(C₁-C₆ alkyl)₂), —N(C₁-C₆        alkyl)((C₁-C₆ alkylene)-CN), —N(C₁-C₆ alkyl)((C₁-C₆        alkylene)N(C₁-C₆ alkyl)₂), —NH(C₁-C₆ alkylene)-O—(C₁-C₆ alkyl),

-   -   a is an integer ranging from 0 to 10;    -   b is an integer ranging from 0 to 8;    -   c is an integer ranging from 0 to 6; and    -   R₅ is

In certain embodiments, Hal is —Cl. In yet another embodiment, x and yare 0.

In certain embodiments, x and y are 0, x is 0 and y is 1, x is 1 and yis 2, x is 1 and y is 0, x is 1 and y is 1, x is 1 and y is 2, x is 2and y is 0, x is 2 and y is 1, or x is 2 and y is 2.

In certain embodiments, Hal is —Cl and: x and y are 0, x is 0 and y is1, x is 1 and y is 2, x is 1 and y is 0, x is 1 and y is 1, x is 1 and yis 2, x is 2 and y is 0, x is 2 and y is 1, or x is 2 and y is 2.

In particular embodiments, x is 1 and R₁ is in the ortho positionrelative to the pyrazolopyridazino ring system. In certain embodiments,x is 1 and R₁ is in the para position relative pyrazolopyridazino ringsystem. In further embodiments, x is 1 and R₁ is in the meta positionrelative pyrazolopyridazino ring system.

In particular embodiments, y is 1 and R₂ is in the ortho positionrelative pyrazolopyridazino ring system. In certain embodiments, y is 1and R₂ is in the para position relative pyrazolopyridazino ring system.In further embodiments, y is 1 and R₂ is in the meta position relativepyrazolopyridazino ring system.

In particular embodiments, x is 2 and R₁ is in the ortho and metaposition relative pyrazolopyridazino ring system. In certainembodiments, x is 2 and R₁ is in the ortho and para position relativepyrazolopyridazino ring system. In further embodiments, x is 2 and R₁ isin the para and meta position relative pyrazolopyridazino ring system.

In particular embodiments, y is 2 and R₂ is in the ortho and metaposition relative pyrazolopyridazino ring system. In certainembodiments, y is 2 and R₂ is in the ortho and para position relativepyrazolopyridazino ring system. In further embodiments, y is 2 and R₂ isin the para and meta position relative pyrazolopyridazino ring system.

In yet other embodiments, R₁ is chloro. In certain embodiments, R₁ isfluoro. In certain embodiments, R₁ is iodo. In other embodiments, R₁ is—Br. In further embodiments, R₁ is —OCH₃. In other embodiments, R₁ is—CH₃. In yet other embodiments, R₁ is —C(O)N(H)CH₃. In certainembodiments, R₁ is —CF₃. In further embodiments, R₁ is —CN. Inadditional embodiments, R₁ is —C≡CCH₂OH.

In yet other embodiments, x is 1 or 2, and R₁ is —Cl, —F, —I, —Br,—OCH₃, —CH₃, —C(O)N(H)CH₃, —CF₃, —CN or —C≡CCH₂OH.

In yet other embodiments, Hal is —Cl, x is 1 or 2, and R₁ is —Cl, —F,—I, —Br, —OCH₃, —CH₃, —C(O)N(H)CH₃, —CF₃, —CN or —C≡CCH₂OH.

In yet other embodiments, R₂ is —Cl. In certain embodiments, R₂ is —F.In other embodiments, R₂ is —Br. In further embodiments, R₂ is —OCH₃. Inother embodiments, R₂ is —CH₃. In yet other embodiments, R₂ is—C(O)N(H)CH₃. In certain embodiments, R₂ is —CF₃. In furtherembodiments, R₂ is —CN. In additional embodiments, R₂ is —C≡CCH₂OH.

In yet other embodiments, y is 1 or 2, and R₂ is —Cl, —F, —Br, —OCH₃,—CH₃, —C(O)N(H)CH₃, —CF₃, —CN or —C≡CCH₂OH.

In yet other embodiments, Hal is —Cl, y is 1 or 2, and R₂ is —Cl, —F,—Br, —OCH₃, —CH₃, —C(O)N(H)CH₃, —CF₃, —CN or —C≡CCH₂OH.

In particular embodiments, R₃ is —H. In certain embodiments, R₃ is —CH₃.In further embodiments, R₃ is —CH₂CH₃. In still further embodiments, R₃is —CHCH₂. In other embodiments, R₃ is —CH₂CH₂OH. In particularembodiments, R₃ is —(CH₂)₂C₆H₅. In other embodiments, R₃ is —CH₂C(O)OH.In yet other embodiments, R₃ is —CH₂C(O)N(H)CH₃. In certain embodiments,R₃ is —CH₂C(O)N(H)((CH₂)₂N(CH₃)₂).

In yet other embodiments, R₃ is —CH₂C(O)N(H)((CH₂)₃N(CH₃)₂). In otherembodiments, R₃ is —CH₂C(O)N(CH₃)CH₂CN. In particular embodiments, R₃ is—CH₂C(O)NH₂. In certain embodiments, R₃ is —CH₂C(O)N(H)((CH₂)₂OH). Inother embodiments,

R₃ is —CH₂C(O)N(H)((CH₂)₂OCH₃). In still further embodiments, R₃ is—CH₂C(CH₃)₂OH.

In yet other embodiments, R₃ is —CH₂C(O)OCH₃. In further embodiments, R₃is —CH₂CH(OH)CH₃. In still further embodiments, R₃ is —CH₂CH₂OH. Inparticular embodiments,

R₃ is —CH(CH₃)CH₂OH.

In further embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In other embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In particular embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In yet other embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In certain embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In other embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In particular embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In yet other embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In certain embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In other embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In particular embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In yet other embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In certain embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In other embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In particular embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In yet other embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In certain embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In other embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In particular embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In yet other embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In certain embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In other embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In particular embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In yet other embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In certain embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In other embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In further embodiments of the invention, R₃ is —CH₂C(O)R₄ and R₄ is

In certain embodiments of the invention, R₃ is —CH₂C(O)R₄ and R₄ is

In other embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In further embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In further embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In particular embodiments, R₃ is —(CH₂)₂R₅ and R₅ is

In yet other embodiments, R₃ is —(CH₂)₂R₅ and R₅ is

In certain embodiments, R₃ is —(CH₂)₂R₅ and R₅ is

In other embodiments, invention, R₃ is —(CH₂)₂R₅ and R₅ is

In some embodiments, a is an integer ranging from 0 to 5. In someembodiments, b is an integer ranging from 0 to 4. In some embodiments, cis an integer ranging from 0 to 6.

Compounds of Formula III

The invention additionally provides compounds of Formula III:

and pharmaceutically acceptable salts thereof,

-   -   wherein Hal is —Cl, —F, —I, or —Br;    -   x is an integer ranging from 0 to 5;    -   each R₁ is independently —Cl, —F, —I, —Br, —C₁-C₃ alkyl,        —O—C₁-C₃ alkyl, —CN, —CF₃, —C(O)NH(CH₃), or —C≡CCH₂OH;    -   R₃ is —H, —C₁-C₆ alkyl, alkylene)-OH, alkylene)-phenyl,        alkylene)-O—(C₁-C₆ alkyl), —C₂-C₆ alkenyl, —(C₁-C₆        alkylene)-C(O)R₄, —(C₁-C₆ alkylene)-R₅,

-   -   R₄ is —OH, —O—(C₁-C₆ alkyl), —NH₂, —NH(C₁-C₆ alkyl), —NH((C₁-C₆        alkylene)-OH), —NH((C₁-C₆ alkylene)N(C₁-C₆ alkyl)₂), —N(C₁-C₆        alkyl)((C₁-C₆ alkylene)-CN), —N(C₁-C₆ alkyl)((C₁-C₆        alkylene)N(C₁-C₆ alkyl)₂), —NH(C₁-C₆ alkylene)-O—(C₁-C₆        alkyl),

-   -   a is an integer ranging from 0 to 10;    -   b is an integer ranging from 0 to 8;    -   c is an integer ranging from 0 to 6;    -   R₅ is

-   -   wherein each R₆ and R₇ is independently —H or —I, wherein at        least one of R₆ and R₇ is —I,    -   and wherein when R₃ is —C₁-C₃ alkyl, R₇ is —H.

In certain embodiments, one R₆ in the ortho position relative to thepyrazolopyridazino ring system is iodo and the remaining R₆ and R₇groups are hydrogen. In other embodiments, one R₆ in the para positionrelative to the pyrazolopyridazino ring system is iodo and the remainingR₆ and R₇ groups are hydrogen. In further embodiments, one R₆ in theortho position relative to the pyrazolopyridazino ring system and one R₆in the para position relative to the pyrazolopyridazino ring system areiodo and the remaining R₆ and R₇ groups are hydrogen. In furtherembodiments, the two R₆ groups in the ortho positions relative to thepyrazolopyridazino ring system and one R₆ in the para position relativeto the pyrazolopyridazino ring system are iodo and the remaining R₆ andR₇ groups are hydrogen. In further embodiments, the two R₆ groups in thepara positions relative to the pyrazolopyridazino ring system and one R₆in the ortho position relative to the pyrazolopyridazino ring system areiodo and the remaining R₆ and R₇ are hydrogen. In certain embodiments,all R₆ groups are iodo and R₇ is hydrogen. In yet further embodiments,R₇ is iodo and the R₆ groups are hydrogen.

In a particular embodiment, one R₆ in the para position relative to thepyrazolopyridazino ring system is iodo and R₃ is —CH₃.

In certain embodiments, Hal is —Cl. In yet another embodiment, x is 0.In another embodiment, x is 1. In a certain embodiments, x is 2.

In particular embodiments, x is 1 and R₁ is in the ortho positionrelative to the pyrazolopyridazino ring system. In certain embodiments,x is 1 and R₁ is in the para position relative pyrazolopyridazino ringsystem. In further embodiments, x is 1 and R₁ is in the meta positionrelative pyrazolopyridazino ring system.

In particular embodiments, x is 2 and R₁ is in the ortho and metaposition relative pyrazolopyridazino ring system. In certainembodiments, x is 2 and R₁ is in the ortho and para position relativepyrazolopyridazino ring system. In further embodiments, x is 2 and R₁ isin the para and meta position relative pyrazolopyridazino ring system.

In yet other embodiments, R₁ is —Cl. In certain embodiments, R₁ is —F.In certain embodiments, R₁ is —I. In further embodiments, R₁ is —OCH₃.In other embodiments, R₁ is —CH₃. In yet other embodiments, R₁ is—C(O)N(H)CH₃. In certain embodiments, R₁ is —CF₃. In furtherembodiments, R₁ is —CN. In additional embodiments, R₁ is —C≡CCH₂OH.

In yet other embodiments, x is 1 or 2, and R₁ is —Cl, —F, —Br, —I,—OCH₃, —CH₃, —C(O)N(H)CH₃, —CF₃, —CN or —C≡CCH₂OH.

In yet other embodiments, Hal is —Cl, x is 1 or 2, and R₁ is —Cl, —F,—Br, —I, —OCH₃, —CH₃, —C(O)N(H)CH₃, —CF₃, —CN or —C≡CCH₂OH.

In particular embodiments, R₃ is —H. In certain embodiments, R₃ is —CH₃.In further embodiments, R₃ is —CH₂CH₃. In still further embodiments, R₃is —CHCH₂. In other embodiments, R₃ is —CH₂CH₂OH. In particularembodiments, R₃ is —(CH₂)₂C₆H₅. In other embodiments, R₃ is —CH₂C(O)OH.In yet other embodiments, R₃ is —CH₂C(O)N(H)CH₃. In certain embodiments,R₃ is —CH₂C(O)N(H)((CH₂)₂N(CH₃)₂). In yet other embodiments, R₃ is—CH₂C(O)N(H)((CH₂)₃N(CH₃)₂). In other embodiments, R₃ is—CH₂C(O)N(CH₃)CH₂CN. In particular embodiments, R₃ is —CH₂C(O)NH₂. Incertain embodiments, R₃ is —CH₂C(O)N(H)((CH₂)₂OH). In other embodiments,R₃ is —CH₂C(O)N(H)((CH₂)₂OCH₃). In still further embodiments, R₃ is—CH₂C(CH₃)₂OH. In yet other embodiments, R₃ is —CH₂C(O)OCH₃. In furtherembodiments, R₃ is —CH₂CH(OH)CH₃. In still further embodiments, R₃ is—CH₂CH₂OH. In particular embodiments, R₃ is —CH(CH₃)CH₂OH.

In further embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In other embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In particular embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In yet other embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In certain embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In other embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In particular embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In yet other embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In certain embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In other embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In particular embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In yet other embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In certain embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In other embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In particular embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In yet other embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In certain embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In other embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In particular embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In yet other embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In certain embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In other embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In particular embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In yet other embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In certain embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In other embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In further embodiments of the invention, R₃ is —CH₂C(O)R₄ and R₄ is

In certain embodiments of the invention, R₃ is —CH₂C(O)R₄ and R₄ is

In other embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In further embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In further embodiments, R₃ is —CH₂C(O)R₄ and R₄ is

In particular embodiments, R₃ is —(CH₂)₂R₅ and R₅ is

In yet other embodiments, R₃ is —(CH₂)₂R₅ and R₅ is

In certain embodiments, R₃ is —(CH₂)₂R₅ and R₅ is

In other embodiments, invention, R₃ is —(CH₂)₂R₅ and R₅ is

In some embodiments, a is an integer ranging from 0 to 5. In someembodiments, b is an integer ranging from 0 to 4. In some embodiments, cis an integer ranging from 0 to 6.

Illustrative Pyrazolopyridazine Compounds

Non-limiting examples of specific Pyrazolopyridazine compounds include:

and pharmaceutically acceptable salts thereof.

Compounds of Formula IV

The invention additionally provides compounds of Formula IV:

and salts thereof,

wherein R₈ is —C₁-C₃ alkyl.

In certain embodiments of the invention, R₈ is —CH₃, in yet furtherembodiments of the invention, R₈ is —CH₂CH₃. In other embodiments of theinvention, R₈ is —CH₂CH₂CH₃. In other embodiments of the invention, R₈is —CH(CH₃)₂.

The invention still further provides Compound 43, which has thestructure:

and salts thereof. Such salts are the reaction product of Compound 43and an inorganic or organic acid. In one embodiment a salt is apharmaceutically acceptable salt.

Compounds of Formula IV, compound 43 and salts thereof are useful asintermediates for synthesizing for Pyrazolopyridazine compounds.

The invention still further provides Compound 44, which has thestructure:

and salts thereof. Such salts are the reaction product of Compound 44and an inorganic or organic acid. In one embodiment a salt is apharmaceutically acceptable salt.

Compound 44 is useful as a probe for identifying proteins that bind toits bisphenyl pyrazolopyridazine moiety.

The invention still further provides Compound 46, which has thestructure:

and pharmaceutically acceptable salts thereof.

Some of the compounds disclosed herein, for example, Compounds 44, 63,72, 74, 83, 88 and 89, are depicted having a bold or hatched wedge,indicating absolute stereochemistry.

Without being bound by any particular mechanism, it is believed that thebisphenyl pyrazolopyridazine moiety of Pyrazolopyridazine compounds isinvolved in the restoration of the activity and trafficking of Clarin I,which is the protein encoded by the gene mutated in Usher III Syndrome(Adato et al., Eur J Hum Genet. 2002 June; 10(6):339-50)

Proteins or domains thereof that can interact with Clarin I, andtherefore can bind to the bisphenyl pyrazolopyridazine moiety ofCompound 44, include, but are not limited to the proteins listed inTable 1 (Tian et al., J Biol Chem. 2009 Jul. 10; 284(28):18980-93):

TABLE 1 4F2 cell-surface antigen heavy chain 78-kDa glucose-regulatedprotein ATP synthase subunit γ, mitochondrial Basigin N-cadherinCalnexin Carboxypeptidase D Catenin a-1 Cation-independentmannose-6-phosphate receptor CD166 antigen CD276 antigen Cell adhesionmolecule 1 Clarin-1 Coxsackievirus and adenovirus receptor Guaninenucleotide-binding protein G(i), a-2 subunit Guanine nucleotide-bindingprotein G(I)/G(S)/G(T) subunit beta-1 Heat shock 70-kDa protein 1Integrin α-5 Integrin α-6 Integrin α-V Integrin β-1 Junctional adhesionmolecule A Junctional adhesion molecule C Monocarboxylate transporter 1Myelin protein zero-like protein 1 Myosin light polypeptide 6Neuropilin-1 Neutral amino acid transporter B(0) Plasma membranecalcium-transporting ATPase 1 Plasma membrane calcium-transportingATPase 4 Prostaglandin F2 receptor negative regulator Protein 4.1Ras-related C3 botulinum toxin substrate 1 Ras-related protein Rab-14Ras-related protein Rab-9A Ras-related protein Ral-A Ras-related proteinRap-1A Secretory carrier-associated membrane protein 1 Secretorycarrier-associated membrane protein 3 Sodium/potassium-transportingATPase subunit a-1 Sodium/potassium-transporting ATPase subunit β-3Solute carrier family 12 member 2 Syntaxin-12 Syntaxin-4 Syntaxin-6Syntaxin-7 Transferrin receptor protein 1 Tumor-associated calciumsignal transducer 1 Type-1 angiotensin II receptor-associated proteinTyrosine-protein kinase-like 7 Vesicle-associated membrane protein 3Voltage-dependent anion-selective channel protein 1 Voltage-dependentanion-selective channel protein 2 Zinc transporter ZIP14

The compounds of the invention can be in the form of a salt. In someembodiments, the salt is a pharmaceutically acceptable salt.Pharmaceutically acceptable salts include, for example, acid-additionsalts and base-addition salts. The acid that forms an acid-addition saltcan be an organic acid or an inorganic acid. A base that forms abase-addition salt can be an organic base or an inorganic base. In someembodiments, a pharmaceutically acceptable salt is a metal salt. In someembodiments, a pharmaceutically acceptable salt is an ammonium salt.

Acid-addition salts can arise from the addition of an acid to thefree-base form of a compound of the invention. In some embodiments, theacid is organic. In some embodiments, the acid is inorganic.Non-limiting examples of suitable acids include hydrochloric acid,hydrobromic acid, hydroiodic acid, nitric acid, nitrous acid, sulfuricacid, sulfurous acid, a phosphoric acid, nicotinic acid, isonicotinicacid, lactic acid, salicylic acid, 4-aminosalicylic acid, tartaric acid,ascorbic acid, gentisinic acid, gluconic acid, glucaronic acid, saccaricacid, formic acid, benzoic acid, glutamic acid, pantothenic acid, aceticacid, propionic acid, butyric acid, fumaric acid, succinic acid, citricacid, oxalic acid, maleic acid, hydroxymaleic acid, methylmaleic acid,glycolic acid, malic acid, cinnamic acid, mandelic acid,2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid, phenylaceticacid, N-cyclohexylsulfamic acid, methanesulfonic acid, ethanesulfonicacid, benzenesulfonic acid, p-toluenesulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,4-methylbenzenesulfonic acid, naphthalene-2-sulfonic acid,naphthalene-1,5-disulfonic acid, 2-phosphoglyceric acid,3-phosphoglyceric acid, glucose-6-phosphoric acid, and an amino acid.

Non-limiting examples of suitable acid-addition salts include ahydrochloride salt, a hydrobromide salt, a hydroiodide salt, a nitratesalt, a nitrite salt, a sulfate salt, a sulfite salt, a phosphate salt,a hydrogen phosphate salt, a dihydrogen phosphate salt, a carbonatesalt, a bicarbonate salt, a nicotinate salt, an isonicotinate salt, alactate salt, a salicylate salt, a 4-aminosalicylate salt, a tartratesalt, an ascorbate salt, a gentisinate salt, a gluconate salt, aglucaronate salt, a saccarate salt, a formate salt, a benzoate salt, aglutamate salt, a pantothenate salt, an acetate salt, a propionate salt,a butyrate salt, a fumarate salt, a succinate salt, a citrate salt, anoxalate salt, a maleate salt, a hydroxymaleate salt, a methylmaleatesalt, a glycolate salt, a malate salt, a cinnamate salt, a mandelatesalt, a 2-phenoxybenzoate salt, a 2-acetoxybenzoate salt, an embonatesalt, a phenylacetate salt, an N-cyclohexylsulfamate salt, amethanesulfonate salt, an ethanesulfonate salt, a benzenesulfonate salt,a p-toluenesulfonate salt, a 2-hydroxyethanesulfonate salt, anethane-1,2-disulfonate salt, a 4-methylbenzenesulfonate salt, anaphthalene-2-sulfonate salt, a naphthalene-1,5-disulfonate salt, a2-phosphoglycerate salt, a 3-phosphoglycerate salt, aglucose-6-phosphate salt, and an amino acid salt.

Metal salts can arise from the addition of an inorganic base to acompound of the invention having a carboxyl group. The inorganic baseconsists of a metal cation paired with a basic couterion, such as, forexample, hydroxide, carbonate, bicarbonate, or phosphate. The metal canbe an alkali metal, alkaline earth metal, transition metal, or maingroup metal. Non-limiting examples of suitable metals include lithium,sodium, potassium, cesium, cerium, magnesium, manganese, iron, calcium,strontium, cobalt, titanium, aluminum, copper, cadmium, and zinc.

Non-limiting examples of suitable metal salts include a lithium salt, asodium salt, a potassium salt, a cesium salt, a cerium salt, a magnesiumsalt, a manganese salt, an iron salt, a calcium salt, a strontium salt,a cobalt salt, a titanium salt, a aluminum salt, a copper salt, acadmium salt, and a zinc salt.

Ammonium salts can arise from the addition of ammonia or an organicamine to a compound of the invention having a carboxyl group.Non-limiting examples of suitable organic amines include triethyl amine,diisopropyl amine, ethanol amine, diethanol amine, triethanol amine,morpholine, N-methylmorpholine, piperidine, N-methylpiperidine,N-ethylpiperidine, dibenzyl amine, piperazine, pyridine, pyrrazole,imidazole, pyrazine, pipyrazine, ethylenediamine, N,N′-dibenzylethylenediamine, procaine, chloroprocaine, choline, dicyclohexyl amine, andN-methylglucamine.

Non-limiting examples of suitable ammonium salts include is atriethylammonium salt, a diisopropylammonium salt, an ethanolammoniumsalt, a diethanolammonium salt, a triethanolammonium salt, amorpholinium salt, an N-methylmorpholinium salt, a piperidinium salt, anN-methylpiperidinium salt, an N-ethylpiperidinium salt, adibenzylammonium salt, a piperazinium salt, a pyridinium salt, apyrrazolium salt, an imidazolium salt, a pyrazinium salt, anethylenediammonium salt, an N,N′-dibenzylethylenediammonium salt, aprocaine salt, a chloroprocaine salt, a choline salt, adicyclohexylammonium salt, and a N-methylglucamine salt.

Methods for Making the Pyrazolopyridazine Compounds

Non-limiting examples of synthetic schema that are useful forsynthesizing the Pyrazolopyridazine compounds include the following.

Scheme 1 generally describes the preparation of Pyrazolopyridazinecompounds having a 1-N-methyl group and where R′ and R″ areindependently an unsubstituted or a substituted phenyl group. Forexample, a 2-cyanocarbonyl compound in which R′ is unsubstituted orsubstituted phenyl is condensed with N-methylhydrazine to provide a3-substituted-1-methyl-1H-pyrazol-5-amine. The 5-amino group isacylated, for example, with acetic anhydride in the presence of a base,such as pyridine, to provide a 5-amido compound. The 5-amido compound isiodinated, for example, with a mixture of iodine and iodic acid in asolvent such as ethanol (EtOH) to provide anN-(3-substituted-4-iodo-1-methyl-1H-pyrazol-5-yl)acetamide. Apalladium-mediated cross-coupling, such as a Sonagashira cross-coupling,of the acetamide with an R″-substituted terminal alkyne, catalyzed, forexample, by a palladium complex such as palladium (II)bistriphenylphosphine dichloride in the presence of copper (I) iodide ina solvent such as dimethylformamide (DMF) with a base such astriethylamine provides a disubstituted alkyne in which R″ isunsubstituted or substituted phenyl. Saponification of the alkyneacetamide with a base such as sodium hydroxide in a solvent such asethanol provides the primary amine. Diazotization of the primary aminewith sodium nitrite in concentrated hydrochloric acid provides a diazointermediate, which cyclizes to provide a Pyrazolopyridazine compoundhaving a 1-N-methyl group and where R′ and R″ are independently anunsubstituted or a substituted phenyl group.

Scheme 2 generally describes the preparation of Pyrazolopyridazinecompounds having an R₃ group and in which R′ is an unsubstituted or asubstituted phenyl group. R′ and R₃ can be the same or different. Forexample, 4,6-dichloro-3-phenylpyridazine is deprotonated with a basesuch as lithium diisopropyl amide (LDA) in a solvent such astetrahydrofuran (THF), and the resultant 5-lithio species is condensedwith an unsubstituted or a substituted benzaldehyde to provide asecondary alcohol. The alcohol is oxidized to a ketone with an oxidizingagent such as manganese dioxide in a solvent such as toluene. The ketoneis condensed with an R₃-substituted hydrazine in a solvent such asethanol to provide an intermediate hydrazone, which cyclizes to providea Pyrazolopyridazine compound having a 1-N—R₃ group, in which R₃ isdefined as in Formulas II and III and in which R′ is an unsubstituted ora substituted phenyl group.

Scheme 3 generally describes the preparation of Pyrazolopyridazinecompounds having a 1-N-methyl group and where R′ is a cyano group, analkyne, an alkene or an aryl group. For example,1-methyl-3-iodophenyl-4-chloro-5-phenyl-1H-pyrazolo[3,4-c]pyridazine iscoupled with a suitable coupling partner, such as a cyanide salt, aterminal alkyne, an alkenyl halide, or an aryl halide, optionally in thepresence of a suitable catalyst such as a palladium complex, optionallyin the presence of a non-palladium transition metal salt such as a zincor copper salt, optionally in the presence of an additive such astriphenylphosphine or an organic amine base, to provide aPyrazolopyridazine compound having a 1-N-methyl group and where R′ is acyano group, an alkyne, an alkene or an aryl group. The position of R′,i.e., ortho, meta or para, in the product is the same as the position ofthe iodo group in the starting material.

Therapeutic Uses

A compound of the invention can be administered to a subject in needthereof for the treatment of a retinal degenerative disease.Non-limiting examples of retinal degenerative diseases include:retinitis pigmentosa, Leber's congenital Amaurosis, Syndromic retinaldegenerations, age-related macular degeneration including wet and dryage-related macular degeneration, and Usher Syndrome. In someembodiments, the Usher Syndrome is a subtype of Usher Syndrome. In someembodiments, the subtype is Usher I. In some embodiments, the subtype isUsher II. In some embodiments, the subtype is Usher III.

In a further embodiment of the invention, a compound of the inventioncan be administered to a subject in need thereof for the treatment ofhearing loss associated with Usher Syndrome. In some embodiments, theUsher Syndrome is a subtype of Usher Syndrome. In some embodiments, thesubtype is Usher I. In some embodiments, the subtype is Usher II. Insome embodiments, the subtype is Usher III.

A “subject” is a mammal, e.g., a human, mouse, rat, guinea pig, dog,cat, horse, cow, pig, or non-human primate, such as a monkey,chimpanzee, baboon or rhesus. In one embodiment, the subject is a human.

The compounds of the invention can be administered to a subject as acomponent of a composition that comprises a pharmaceutically acceptablecarrier or vehicle. Non-limiting examples of suitable pharmaceuticalcarriers or vehicles include starch, glucose, lactose, sucrose, gelatin,malt, rice, flour, chalk, silica gel, magnesium carbonate, magnesiumstearate, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol, bufferedwater, and phosphate buffered saline. These compositions can beadministered as, for example, drops, solutions, suspensions, tablets,pills, capsules, powders, and sustained-release formulations. In someembodiments, the compositions comprise, for example, lactose, dextrose,sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate,alginates, tragacanth, gelatin, calcium silicate, microcrystallinecellulose, polyvinylpyrrolidone, cellulose, water syrup, methylcellulose, methyl and propylhydroxybenzoates, talc, magnesium stearate,and mineral oil. The compositions can additionally comprise lubricatingagents, wetting agents, emulsifying and suspending agents, preservingagents, sweetening agents or flavoring agents.

The compositions can comprise an effective amount of a compound of theinvention. An “effective amount” of a compound of the invention is anamount that is effective to treat a retinal degenerative disease orhearing loss associated with Usher Syndrome in a subject. Thecompositions can be formulated in a unit dosage form that comprises aneffective amount of a compound of the invention. In some embodiments,the compositions comprise, for example, from about 1 ng to about 1,000mg of a compound of the invention. In some embodiments, the compositionscomprise from about 100 mg to about 1,000 mg of a compound of theinvention. In some embodiments, the compositions comprise from about 100mg to about 500 mg of a compound of the invention. In some embodiments,the compositions comprise from about 200 mg to about 300 mg of acompound of the invention.

The dosage of a compound of the invention can vary depending on thesymptoms, age, and body weight of the subject, the nature and severityof the retinal degenerative disease or hearing loss associated withUsher Syndrome, the route of administration, and the form of thecomposition. The compositions described herein can be administered in asingle dose or in divided doses. In some embodiments, the dosage of acompound of the invention ranges from about 0.01 ng to about 10 g per kgbody mass of the subject, from about 1 ng to about 0.1 g per kg, or fromabout 100 ng to about 10 mg per kg.

Administration can be, for example, topical, intraaural, intraocular,parenteral, intravenous, intra-arterial, subcutaneous, intramuscular,intracranial, intraorbital, intraventricular, intracapsular,intraspinal, intracisternal, intraperitoneal, intranasal, aerosol,suppository, or oral. Formulations for oral use include tabletscontaining a compound of the invention in a mixture with non-toxicpharmaceutically acceptable excipients. These excipients can be, forexample, inert diluents or fillers (e.g., sucrose and sorbitol),lubricating agents, glidants, and antiadhesives (e.g., magnesiumstearate, zinc stearate, stearic acid, silicas, hydrogenated vegetableoils, or talc). Formulations for ocular use can be in the form ofeyedrops.

A compound of the invention or composition thereof can be provided inlyophilized form for reconstituting, for instance, in isotonic, aqueous,or saline buffers for parental, subcutaneous, intradermal,intramuscular, or intravenous administration. A composition of theinvention can also be in the form of a liquid preparation useful fororal, intraaural, nasal, or sublingual administration, such as asuspension, syrup or elixir. A composition of the invention can also bein a form suitable for oral administration, such as a capsule, tablet,pill, and chewable solid formulation. A composition of the invention canalso be prepared as a cream for dermal administration as a liquid, aviscous liquid, a paste, or a powder. A composition of the invention canalso be prepared as a powder for pulmonary administration with orwithout an aerosolizing component.

The compositions can be in oral, intraaural, intranasal, sublingual,intraduodenal, subcutaneous, buccal, intracolonic, rectal, vaginal,mucosal, pulmonary, transdermal, intradermal, parenteral, intravenous,intramuscular and ocular dosage forms as well as being able to traversethe blood-brain barrier.

The compositions of the invention can be administered by various meansknown in the art. For example, the compositions of the invention can beadministered orally, and can be formulated as tablets, capsules,granules, powders or syrups. Alternatively, compositions of theinvention can be administered parenterally as injections (for example,intravenous, intramuscular or subcutaneous), drop infusion preparationsor suppositories. For ophthalmic application compositions of theinvention can be formulated as eye drops or eye ointments. Auralcompositions can be formulated as ear drops, ointments, creams, liquids,gels, or salves for application to the ear, either internally orsuperficially. These formulations can be prepared by conventional means,and the compositions can be mixed with any conventional additive, suchas an excipient, a binder, a disintegrating agent, a lubricant, asolubilizing agent, a suspension aid, an emulsifying agent, or a coatingagent.

Compositions of the invention can include wetting agents, emulsifiers,and lubricants, coloring agents, release agents, coating agents,sweetening, flavoring and perfuming agents, preservatives andantioxidants.

Compositions can be suitable, for example, for oral, intraaural,intraocular, nasal, topical (including buccal and sublingual), rectal,vaginal, aerosol and/or parenteral administration. The compositions canbe provided in a unit dosage form, and can be prepared by any methodsknown in the art.

Formulations suitable for oral administration may be in the form ofcapsules, cachets, pills, tablets, lozenges, powders, granules, or as asolution or a suspension in an aqueous or non-aqueous liquid, or as anoil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup,or as pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia. Compositions of the invention can also beadministered as a bolus, electuary, or paste.

Additional examples of pharmaceutically acceptable carriers or vehiclesinclude: (1) fillers or extenders, such as starches, lactose, sucrose,glucose, mannitol, and/or silicic acid; (2) binders, such ascarboxymethyl cellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; (3) humectants, such as glycerol; (4)disintegrating agents, such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, and sodium carbonate;(5) solution retarding agents, such as paraffin; (6) absorptionaccelerators, such as quaternary ammonium compounds; (7) wetting agents,such as acetyl alcohol and glycerol monostearate; (8) absorbents, suchas kaolin and bentonite clay; (9) lubricants, such as talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, and mixtures thereof; (10) coloring agents; and (11) bufferingagents. Similar compositions can be employed as fillers in soft- orhard-filled gelatin capsules.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, gels, solutions, suspensions,syrups and elixirs. The liquid dosage form can contain inert diluentscommonly used in the art, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, diethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils such as,cottonseed, groundnut, corn, germ, olive, castor and sesame oils,glycerol, tetrahydrofuryl alcohol, polyethylene glycols, fatty acidesters of sorbitan, and mixtures thereof.

Suspension dosage forms can contain suspending, for example, ethoxylatedisostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters,microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agarand tragacanth, and mixtures thereof.

The dosage forms for transdermal administration of a subject compositioninclude drops, powders, sprays, ointments, pastes, creams, lotions,gels, solutions, and patches. The ointments, pastes, creams, and gelscan contain excipients, such as animal and vegetable fats, oils, waxes,paraffin, starch, tragacanth, cellulose derivatives, polyethyleneglycols, silicones, bentonite, silicic acid, talc and zinc oxide, ormixtures thereof.

Powders and sprays can contain excipients such as lactose, talc, silicicacid, aluminum hydroxide, calcium silicates, polyamide powder, ormixtures thereof. Sprays may additionally contain customary propellants,such as chlorofluorohydrocarbons and volatile unsubstitutedhydrocarbons, such as butane and propane.

Compositions can be administered by aerosol of solid particles. Anon-aqueous (e.g., fluorocarbon propellant) suspension could be used.Sonic nebulizers can be used because they minimize exposure to shear,which might cause degradation.

An aqueous aerosol can be made by formulating an aqueous solution orsuspension of a compound of the invention with any conventionalpharmaceutically acceptable carriers or vehicles such non-ionicsurfactants (Tweens, Pluronics, or polyethylene glycol); proteins suchas serum albumin; sorbitan esters; fatty acids; lecithin; amino acids;buffers; salts; sugars; or sugar alcohols.

Compositions suitable for parenteral administration comprise a compoundof the invention and one or more pharmaceutically acceptable sterileisotonic aqueous or non-aqueous solutions, dispersions, suspensions, oremulsions, or sterile powders which can be reconstituted into sterileinjectable solutions or dispersions just prior to use, which can containantioxidants, buffers, bacteriostats, or solutes, which render theformulation isotonic with the blood of the subject, and suspending orthickening agents.

Having described the invention with reference to certain embodiments,other embodiments will become apparent to one skilled in the art fromconsideration of the specification. The invention is further defined byreference to the following examples. It will be apparent to thoseskilled in the art that many modifications, both to materials andmethods, may be practiced without departing from the scope of theinvention.

EXAMPLES Example 14-chloro-3-(2-chlorophenyl)-1-methyl-5-phenyl-1H-pyrazolo[3,4-c]pyridazine(Compound 37)

Step 1: 3-(2-chlorophenyl)-1-methyl-1H-pyrazol-5-amine

A mixture of 2-chlorobenzoylacetonitrile (540 mg, 3 mmol) andmethylhydrazine (158 μl, 3 mmol) in methanol (3 mL) was heated at 120°C. for 1 hour in a microwave. The reaction mixture was concentrated invacuo to give 3-(2-chlorophenyl)-1-methyl-1H-pyrazol-5-amine as a solid(617 mg), which was used as such in the subsequent step.

Step 2: N-(3-(2-chlorophenyl)-1-methyl-1H-pyrazol-5-yl)acetamide

To a solution of 3-(2-chlorophenyl)-1-methyl-1H-pyrazol-5-amine (617 mg,3 mmol) in pyridine (6 mL) was added acetic anhydride (425 μl, 4.5mmol). The reaction was stirred at room temperature for 16 h. Themixture was concentrated in vacuo. The crude residue was purified bycolumn chromatography (silica gel, gradient 0 to 100% ethylacetate/isohexane) yieldingN-(3-(2-chlorophenyl)-1-methyl-1H-pyrazol-5-yl)acetamide as a solid (526mg).

Step 3: N-(3-(2-chlorophenyl)-4-iodo-1-methyl-1H-pyrazol-5-yl)acetamide

To a solution ofN-(3-(2-chlorophenyl)-1-methyl-1H-pyrazol-5-yl)acetamide (526 mg, 2.12mmol) in ethanol (16 mL) were added iodic acid (93 mg, 0.53 mmol) andiodine (294 mg, 1.16 mmol). The reaction was stirred for 18 h at 50° C.The reaction mixture was concentrated in vacuo and the residue wasdissolved in CH₂Cl₂ and washed with a 2 N Na₂SO₃ solution. The organicphase was dried over MgSO₄, filtered and evaporated to giveN-(3-(2-chlorophenyl)-4-iodo-1-methyl-1H-pyrazol-5-yl)acetamide as alight orange solid (756 mg), which was used as such in the subsequentstep.

Step 4:N-(3-(2-chlorophenyl)-1-methyl-4-(phenylethynyl)-1H-pyrazol-5-yl)acetamide

To a degassed solution of phenylacetylene (157 μl, 1.43 mmol) and Et₃N(3.3 mL) in DMF (1.2 mL) were addedN-(3-(2-chlorophenyl)-4-iodo-1-methyl-1H-pyrazol-5-yl)acetamide (413 mg,1.1 mmol), CuI (42 mg, 0.22 mmol) and PdCl₂(PPh₃)₂ (77 mg, 0.11 mmol).The mixture was flushed with N₂ and then heated to 85° C. for 3 h. Thereaction mixture was concentrated in vacuo and the residue was purifiedby column chromatography (silica gel, gradient 0 to 100% ethylacetate/isohexane) yieldingN-(3-(2-chlorophenyl)-1-methyl-4-(phenylethynyl)-1H-pyrazol-5-yl)acetamideas a solid (181 mg).

Step 5: 3-(2-chlorophenyl)-1-methyl-4-(phenylethynyl)-1H-pyrazol-5-amine

A solution ofN-(3-(2-chlorophenyl)-1-methyl-4-(phenylethynyl)-1H-pyrazol-5-yl)acetamide(170 mg, 0.49 mmol) in ethanol (1 mL) and 25% NaOH (1.4 mL) was heatedto 85° C. for 30 h. The reaction mixture was partitioned between ethylacetate and water. The aqueous phase was washed with ethyl acetate andthe organic phases combined, dried over MgSO₄, filtered and evaporatedto give 3-(2-chlorophenyl)-1-methyl-4-(phenylethynyl)-1H-pyrazol-5-amineas a solid (136 mg), which was used as such in the subsequent step.

Step 6:4-chloro-3-(2-chlorophenyl)-1-methyl-5-phenyl-1H-pyrazolo[3,4-c]pyridazine(Compound 37)

To a cooled (cooling bath −15° C.) stirred suspension of3-(2-chlorophenyl)-1-methyl-4-(phenylethynyl)-1H-pyrazol-5-amine (136mg, 0.44 mmol) in conc. HCl (3.7 mL) was added a solution of sodiumnitrite (34 mg, 0.49 mmol) in water (0.3 mL). After 5 min, the coolingbath was removed and the reaction mixture was stirred at roomtemperature for 1 h. The reaction was cooled again (0° C.) and pouredonto a sat. NaHCO₃ solution. Ethyl acetate was added and pH was adjustedto 7-8 with sat. NaHCO₃. The aqueous phase was extracted with ethylacetate and the organic phases combined, dried over MgSO₄, filtered andevaporated. Crude material was purified by column chromatography (silicagel, gradient 0 to 4% ethyl acetate/CH₂Cl₂) yielding Compound 37 as asolid (47 mg).

LCMS (10 cm_ESCI_Formic) Rt 4.72 min; m/z 355 [M+H] 97.96% purity.

Example 24-chloro-3-(3-methoxyphenyl)-1-methyl-5-phenyl-1H-pyrazolo[3,4-c]pyridazine(Compound 17)

Compound 17 was synthesised following similar procedures outlined inExample 1 (Compound 37), using 3-methoxybenzoylacetonitrile instead of2-chlorobenzoyl acetonitrile in Step 1.

LCMS (10 cm_ESCI_Formic) Rt 4.6 min; m/z 351 [M+H] 96.23% purity.

Example 34-chloro-3-(4-methoxyphenyl)-1-methyl-5-phenyl-1H-pyrazolo[3,4-c]pyridazine(Compound 27)

Compound 27 was synthesised following similar procedures outlined inExample 1 (Compound 37), using 4-methoxybenzoylacetonitrile instead of2-chlorobenzoyl acetonitrile in Step 1.

LCMS (10 cm_ESCI_Bicarb) Rt 3.97 min; m/z 351 [M+H] 96.77% purity.

Example 44-chloro-3-(3-chlorophenyl)-1-methyl-5-phenyl-1H-pyrazolo[3,4-c]pyridazine(Compound 13)

Compound 13 was synthesised following similar procedures outlined inExample 1 (Compound 37), using 3-chlorobenzoylacetonitrile instead of2-chlorobenzoyl acetonitrile in Step 1.

LCMS (10 cm_ESI_Bicarb) Rt 4.16 min; m/z 355 [M+H] 98.38% purity.

Example 54-chloro-3-(3-methoxyphenyl)-5-(4-methoxyphenyl)-1-methyl-1H-pyrazolo[3,4-c]pyridazine(Compound 15)

Compound 15 was synthesised following similar procedures outlined inExample 1 (Compound 37), using 3-methoxybenzoylacetonitrile instead of2-chlorobenzoyl acetonitrile in Step 1 and 4-methoxyphenyl acetyleneinstead of phenyl acetylene in Step 4.

LCMS (10 cm_ESCI_Bicarb) Rt 4.3 min; m/z 381 [M+H] 95.3% purity.

Example 64-chloro-3,5-bis(4-fluorophenyl)-1-methyl-1H-pyrazolo[3,4-c]pyridazine(Compound 33)

Compound 33 was synthesised following similar procedures outlined inExample 1 (Compound 37), using 4-fluorobenzoylacetonitrile instead of2-chlorobenzoyl acetonitrile in Step 1. As a slightly modifiedprocedure, the residue of Step 1 was purified by column chromatography(silica gel, gradient 30 to 100% EtOAc/isohexane). The residue of Step 3was dissolved in CH₂Cl₂ and washed with an aqueous Na₂S₂O₃ solution.Product of Step 3 was purified by column chromatography (silica gel,gradient 0 to 100% ethyl acetate/CH₂Cl₂. 4-fluorophenyl acetylene wasused instead of phenyl acetylene in Step 4. The reaction mixture of Step4 was worked up with CH₂Cl₂, water and K₂CO₃ instead of EtOAc, aq. sat.NaHCO₃. The residue was purified by column chromatography (silica gel,gradient 10 to 100% EtOAc/isohexane) to provide Compound 33.

LCMS (10 cm_ESCI_Formic) Rt 4.18 min; m/z 357 [M+H] 99.63% purity.

Example 7 4-chloro-1-methyl-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazine(Compound 39)

Compound 39 was synthesised following similar procedures outlined inExample 1 (Compound 37), starting from Step 2 using1-methyl-3-phenyl-1H-pyrazol-5-amine instead of3-(2-chlorophenyl)-1-methyl-1H-pyrazol-5-amine.

LCMS (10 cm_ESCI_Bicarb) Rt 4.39 min; m/z 321 [M+H] 95.87% purity.

Example 84-chloro-3-(4-chlorophenyl)-1-methyl-5-phenyl-1H-pyrazolo[3,4-c]pyridazine(Compound 26)

Compound 26 was synthesised following similar procedures outlined inExample 1 (Compound 37), starting from Step 2 using3-(4-chlorophenyl)-1-methyl-1H-pyrazol-5-amine instead of3-(2-chlorophenyl)-1-methyl-1H-pyrazol-5-amine.

LCMS (10 cm_ESCI_Formic) Rt 4.91 min; m/z 355 [M+H] 99.36% purity.

Example 94-chloro-5-(4-fluorophenyl)-1-methyl-3-phenyl-1H-pyrazolo[3,4-c]pyridazine(Compound 4)

Compound 4 was synthesised following similar procedures outlined inExample 1 (Compound 37), starting from Step 2 using1-methyl-3-phenyl-1H-pyrazol-5-amine instead of3-(2-chlorophenyl)-1-methyl-1H-pyrazol-5-amine and4-fluorophenylacetylene instead of phenyl acetylene in Step 4.

LCMS (15 cm_Bicarb_GeminiNX_HPLC) Rt 11.15 min; m/z 339 [M+H] 97.86%purity.

Example 104-Chloro-5-(4-methoxyphenyl)-1-methyl-3-phenyl-1H-pyrazolo[3,4-c]pyridazine(Compound 8)

Compound 8 was synthesised following similar procedures outlined inExample 6 (Compound 33), starting from Step 2 using1-methyl-3-phenyl-1H-pyrazol-5-amine instead of3-(4-fluorophenyl)-1-methyl-1H-pyrazol-5-amine.

LCMS (15 cm_Formic_ASCENTIS_HPLC) Rt 10.61 min; m/z 351 [M+H] 98.07%purity.

Example 114-Chloro-3-(4-fluorophenyl)-1-methyl-5-phenyl-1H-pyrazolo[3,4-c]pyridazine(Compound 14)

Compound 14 was synthesised following similar procedures outlined inExample 1 (Compound 37), starting from Step 2 using3-(4-fluorophenyl)-1-methyl-1H-pyrazol-5-amine instead of3-(2-chlorophenyl)-1-methyl-1H-pyrazol-5-amine.

LCMS (10 cm_ESI_Bicarb) Rt 3.88 min; m/z 339 [M+H] 98.17% purity.

Example 124-Chloro-5-(3-fluorophenyl)-1-methyl-3-phenyl-1H-pyrazolo[3,4-c]pyridazine(Compound 10)

Compound 10 was synthesised following similar procedures outlined inExample 1 (Compound 37), starting from Step 2 using1-methyl-3-phenyl-1H-pyrazol-5-amine instead of3-(2-chlorophenyl)-1-methyl-1H-pyrazol-5-amine and3-fluorophenylacetylene instead of phenyl acetylene in Step 4.

LCMS (10 cm_ESI_Formic) Rt 4.18 min; m/z 339 [M+H] 96.5% purity.

Example 134-Chloro-1-methyl-3-phenyl-5-p-tolyl-1H-pyrazolo[3,4-c]pyridazine(Compound 11)

Compound 11 was synthesised following similar procedures outlined inExample 1 (Compound 37), starting from Step 2 using1-methyl-3-phenyl-1H-pyrazol-5-amine instead of3-(2-chlorophenyl)-1-methyl-1H-pyrazol-5-amine and 4-tolylacetyleneinstead of phenyl acetylene in Step 4.

LCMS (10 cm_ESI_Bicarb) Rt 4.07 min; m/z 335 [M+H] 98.54% purity.

Example 144-Chloro-1-methyl-3-phenyl-5-m-tolyl-1H-pyrazolo[3,4-c]pyridazine(Compound 16)

Compound 16 was synthesised following similar procedures outlined inExample 1 (Compound 37), starting from Step 2 using1-methyl-3-phenyl-1H-pyrazol-5-amine instead of3-(2-chlorophenyl)-1-methyl-1H-pyrazol-5-amine and 3-tolylacetyleneinstead of phenyl acetylene in Step 4.

LCMS (10 cm_ESI_Bicarb) Rt 4.07 min; m/z 335 [M+H] 98.36% purity.

Example 154-chloro-1-methyl-3-phenyl-5-(3-(trifluoromethyl)phenyl)-1H-pyrazolo[3,4-c]pyridazine(Compound 29)

Compound 29 was synthesised following similar procedures outlined inExample 1 (Compound 37), starting from Step 2 using1-methyl-3-phenyl-1H-pyrazol-5-amine instead of3-(2-chlorophenyl)-1-methyl-1H-pyrazol-5-amine and3-(trifluoromethyl)-phenylacetylene instead of phenyl acetylene in Step4.

LCMS (15 cm_Formic_ASCENTIS_HPLC) Rt 13.11 min; m/z 389 [M+H] 86.58%purity.

Example 162-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)-N-methylacetamide(Compound 5)

Compound 5 was synthesised from2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)acetic acid,which was synthesised following similar procedures outlined in Example 1(Compound 37), using benzoylacetonitrile instead of 2-chlorobenzoylacetonitrile and ethyl hydrazinoacetate hydrochloride and 1.5 equivalentof triethylamine instead of methylhydrazine in Step 1. To a suspensionof 2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)acetic acid(73 mg, 0.2 mmol) in CH₂Cl₂ (1 mL) were added 2-hydroxypyridine-N-oxide(HOPO) (22 mg, 0.2 mmol), methylamine hydrochloride (14 mg, 0.2 mmol),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDAC) (50mg, 0.25 mmol), diisopropylethylamine (DIPEA) (90 μl, 0.2 mmol) and DMF(0.5 mL). The reaction mixture was stirred at room temperature for 90min then concentrated in vacuo. The residue was purified by preparativeHPLC yielding Compound 5 as an off-white solid (6 mg).

LCMS (15 cm_Bicarb_GeminiNX_HPLC) Rt 10.02 min; m/z 378 [M+H] 94.61%purity.

Example 172-(4-Chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)-1-(4-methylpiperazin-1-yl)ethanone(Compound 1)

Compound 1 was synthesised from2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)acetic acidfollowing a similar procedure outlined in Example 16 (Compound 5), usingN-methylpiperazine instead of methylamine hydrochloride in the finalstep. The residue in the final step was purified by preparative HPLC,followed by column chromatography (silica gel, gradient 0 to 5%triethylamine/acetone), yielding the Compound 1 as an off-white solid (6mg).

LCMS (15 cm_Bicarb_GeminiNX_HPLC) Rt 10.12 min; m/z 447 [M+H] 96.8%purity.

Example 182-(4-Chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)-N-(2-(dimethylamino)ethyl)acetamide(Compound 2)

Compound 2 was synthesised from2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)acetic acidfollowing a similar procedure outlined in Example 17 (Compound 1), usingN,N′-dimethylethylenediamine instead of N-methylpiperazine in the finalstep.

LCMS (15 cm_Formic_ASCENTIS_HPLC) Rt 7.68 min; m/z 435 [M+H] 95.51%purity.

Example 192-(4-Chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)-1-(4-isopropylpiperazin-1-yl)ethanone(Compound 38)

Compound 38 was synthesised from2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)acetic acidfollowing a similar procedure outlined in Example 17 (Compound 1), usingN-isopropylpiperazine instead of N-methylpiperazine in the final step.

LCMS (15 cm_Formic_ASCENTIS_HPLC) Rt 12.54 min; m/z 475 [M+H] 95.82%purity.

Example 204-(4-Chloro-1-methyl-5-phenyl-1H-pyrazolo[3,4-c]pyridazin-3-yl)-N-methylbenzamide(Compound 25)

Compound 25 was synthesised following similar procedures outlined inExample 1 (Compound 37), using methyl 4-(cyanoacetyl)benzoate instead of2-chlorobenzoyl acetonitrile in Step 1.4-(5-Amino-1-methyl-4-(phenylethynyl)-1H-pyrazol-3-yl)benzoic acidobtained in Step 5 was converted to4-(5-amino-1-methyl-4-(phenylethynyl)-1H-pyrazol-3-yl)-N-methylbenzamidefollowing a similar procedure outlined in Example 16 (Compound 5).4-(5-Amino-1-methyl-4-(phenylethynyl)-1H-pyrazol-3-yl)-N-methylbenzamidewas converted to the Compound 25 following a similar procedure outlinedin Step 5 of Example 5.

LCMS (10 cm_ESI_Formic) Rt 3.1 min; m/z 378 [M+H] 99.89% purity.

Example 212-(4-Chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)-N-(3-(7-nitrobenzo[c][1,2,5]oxadiazol-4-ylamino)propyl)acetamide(Compound 6)

Compound 6 was synthesised from2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)acetic acidfollowing a similar procedure outlined in Example 16 (Compound 5), usingN1-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)propane-1,3-diamine instead ofmethylamine hydrochloride in the final step.

LCMS (10 cm_ESI_Bicarb) Rt 3.59 min; m/z 584 [M+H] 97.74% purity.

Example 22 4-Chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazine (Compound9)

Step 1: (3,5-dichloro-6-phenylpyridazin-4-yl)(phenyl)methanol

To a solution of LDA (2 M in THF, 2.4 mL, 4.7 mmol) in THF (8 mL) at−78° C. was added dropwise a solution of 3-phenyl-4,6-dichloropyridazine(700 mg, 3.1 mmol) in THF (2 mL). After 30 min at −78° C., benzaldehyde(400 mg, 3.75 mmol) was added. The reaction mixture was allowed to warmup to room temperature overnight. The reaction mixture was partitionedbetween ethyl acetate and water. The aqueous phase was extracted withethyl acetate and the organic phases were combined, washed with waterand brine, dried over MgSO₄, filtered and concentrated in vacuo. Thecrude residue was purified by column chromatography (silica gel,gradient 10 to 20% ethyl acetate/isohexane) yielding3,5-dichloro-6-phenylpyridazin-4-yl)(phenyl)methanol as a yellow solid(200 mg).

Step 2: (3,5-dichloro-6-phenylpyridazin-4-yl)(phenyl)methanone

A mixture of (3,5-dichloro-6-phenylpyridazin-4-yl)(phenyl)methanol (200mg, 0.6 mmol), manganese dioxide (700 mg, 8 mmol) in dry toluene (20 mL)was heated at 115° C. for 3 h under Dean Stark conditions. The reactionmixture was cooled and filtered, and the residue was washed with CH₂Cl₂.The filtrate was concentrated in vacuo. The crude residue was purifiedby column chromatography (silica gel, gradient 10 to 20% ethylacetate/isohexane) yielding(3,5-dichloro-6-phenylpyridazin-4-yl)(phenyl)methanone as a solid (150mg).

Step 3: 4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazine (Compound 9)

A mixture of (3,5-dichloro-6-phenylpyridazin-4-yl)(phenyl)methanone (150mg, 0.46 mmol) and hydrazine hydrate (27 mg, 0.55 mmol) in ethanol (0.5mL) was stirred at 40° C. for 1 h in a sealed tube. The reaction mixturewas cooled and filtered, and the residue was washed with diethyl ether.The filtrate was concentrated in vacuo. The crude residue was purifiedby column chromatography (silica gel, gradient 0 to 20% ethylacetate/isohexane) yielding Compound 9 as a white solid (12 mg).

LCMS (10 cm_ESCI_Formic) Rt 4.16 min; m/z 307 [M+H] 95.8% purity.

Example 234-Chloro-3-(4-iodophenyl)-1-methyl-5-phenyl-1H-pyrazolo[3,4-c]pyridazine(Compound 43)

Compound 43 was synthesised following similar procedures outlined inExample 22 (Compound 9), using 4-iodobenzaldehyde instead ofbenzaldehyde in Step 1 and methyl hydrazine instead of hydrazine hydratein Step 3. As a slightly modified procedure, Step 3 was performed at 60°C. for 4 h in a sealed tube. Subsequently, the mixture was cooled andfiltered, and the residue was washed with cold ethanol. The filteredresidue was purified by column chromatography (silica gel, gradient 5 to10% ethyl acetate/isohexane) yielding Compound 43 as an off-white solid.

LCMS (15 cm_Bicarb_GeminiNX_HPLC) Rt 12.63 min; m/z 447 [M+H] 97.4%purity.

Example 244-chloro-1-methyl-5-phenyl-3-m-tolyl-1H-pyrazolo[3,4-c]pyridazine(Compound 19)

Compound 19 was synthesised following similar procedures outlined inExample 22 (Compound 9), using 3-methylbenzaldehyde instead ofbenzaldehyde in Step 1 and methyl hydrazine instead of hydrazine hydratein Step 3. As a slightly modified procedure, Step 3 was performed at 60°C. for 16 h in a sealed tube. Subsequently, the reaction mixture waspartitioned between CH₂Cl₂ and water. The organic phase was separatedusing a phase separating cartridge and concentrated in vacuo. The cruderesidue was purified by column chromatography (silica gel, gradient 5 to20% ethyl acetate/isohexane) yielding Compound 19 as a solid.

LCMS (15 cm_Bicarb_GeminiNX_HPLC) Rt 11.47 min; m/z 335 [M+H] 97.18%purity.

Example 254-Chloro-1-methyl-5-phenyl-3-o-tolyl-1H-pyrazolo[3,4-c]pyridazine(Compound 20)

Compound 20 was synthesised following similar procedures outlined inExample 22 (Compound 9), using 2-methylbenzaldehyde instead ofbenzaldehyde in Step 1 and methyl hydrazine instead of hydrazine hydratein Step 3.

LCMS (10 cm_ESI_Formic) Rt 4.2 min; m/z 335 [M+H] 98.48% purity.

Example 264-Chloro-1-methyl-5-phenyl-3-p-tolyl-1H-pyrazolo[3,4-c]pyridazine(Compound 21)

Compound 21 was synthesised following similar procedures outlined inExample 24 (Compound 19), using 4-methylbenzaldehyde instead of3-methylbenzaldehyde in Step 1. As a slightly modified procedure, Step 3was performed at 70° C. for 16 h in a sealed tube.

LCMS (15 cm_Bicarb_GeminiNX_HPLC) Rt 11.44 min; m/z 335 [M+H] 97.13%purity.

Example 274-Chloro-3-(2-fluorophenyl)-1-methyl-5-phenyl-1H-pyrazolo[3,4-c]pyridazine(Compound 22)

Compound 22 was synthesised following similar procedures outlined inExample 24 (Compound 19), using 2-fluorobenzaldehyde instead of3-methylbenzaldehyde in Step 1.

LCMS (10 cm_ESI_Formic) Rt 4.04 min; m/z 339 [M+H] 96.36% purity.

Example 284-Chloro-3-(3-fluorophenyl)-1-methyl-5-phenyl-1H-pyrazolo[3,4-c]pyridazine(Compound 32)

Compound 32 was synthesised following similar procedures outlined inExample 24 (Compound 19), using 3-fluorobenzaldehyde instead of3-methylbenzaldehyde in Step 1.

LCMS (15 cm_Formic_ASCENTIS_HPLC) Rt 12.79 min; m/z 339 [M+H] 98.58%purity.

Example 294-Chloro-1-methyl-5-phenyl-3-(4-(trifluoromethyl)phenyl)-1H-pyrazolo[3,4-c]pyridazine(Compound 23)

Compound 23 was synthesised following similar procedures outlined inExample 24 (Compound 19), using 4-trifluoromethylbenzaldehyde instead of3-methylbenzaldehyde in Step 1. As a slightly modified procedure, inStep 2, the reaction mixture was filtered and following evaporation ofthe filtrate, purification of(3,5-dichloro-6-phenylpyridazin-4-yl)(4-(trifluoromethyl)phenyl)methanonewas carried out by trituration of the solid residue in diethyl ether.

LCMS (10 cm_ESCI_Bicarb) Rt 4.68 min; m/z 389 [M+H] 98.96% purity.

Example 304-Chloro-3-(3-iodophenyl)-1-methyl-5-phenyl-1H-pyrazolo[3,4-c]pyridazine(Compound 3)

Compound 3 was synthesised following similar procedures outlined inExample 25 (Compound 20), using 3-iodobenzaldehyde instead of2-methylbenzaldehyde in Step 1. As a slightly modified procedure, thecrude residue of Step 3 was purified by column chromatography (silicagel, gradient 80 to 100% CH₂Cl₂/isohexane to 2% diethyl ether/CH₂Cl₂),yielding Compound 3 as a solid.

LCMS (10 cm_ESCI_Formic) Rt 5.03 min; m/z 447 [M+H] 99.13% purity.

Example 31 4-Chloro-1-ethyl-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazine(Compound 18)

Compound 18 was synthesised following similar procedures outlined inExample 22 (Compound 9), using ethyl hydrazine instead of hydrazinehydrate in Step 3. As a slightly modified procedure, Step 3 wasperformed at 90° C. for 16 h in a sealed tube. Subsequently, thereaction mixture was partitioned between CH₂Cl₂ and water. The organicphase was separated using a phase separating cartridge and concentratedin vacuo. The crude residue was purified by column chromatography(silica gel, 5% ethyl acetate/isohexane) yielding Compound 18 as asolid.

LCMS (15 cm_Bicarb_GeminiNX_HPLC) Rt 11.52 min; m/z 335 [M+H] 94.7%purity.

Example 322-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)ethanol(Compound 7)

Compound 7 was synthesised following similar procedures outlined inExample 31 (Compound 18), using 2-hydroxyethyl hydrazine instead ofethyl hydrazine in Step 3. As a slightly modified procedure, Step 3 wasperformed at 90° C. for 30 min in the microwave. The crude residue waspurified by column chromatography (silica gel, gradient 10 to 50% ethylacetate/isohexane), yielding Compound 7 as a solid.

LCMS (15 cm_Bicarb_GeminiNX_HPLC) Rt 10.29 min; m/z 351 [M+H] 95.59%purity.

Example 33 1-Benzyl-4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazine(Compound 31)

Compound 31 was synthesised following similar procedures outlined inExample 31 (Compound 18), using benzyl hydrazine dihydrochloride insteadof ethyl hydrazine in Step 3. As a slightly modified procedure, Step 3was performed at 50° C. for 16 h. The crude residue was purified bycolumn chromatography (silica gel, gradient 5 to 10% ethylacetate/isohexane), followed by preparative HPLC, yielding Compound 31as a solid.

LCMS (15 cm_Formic_ASCENTIS_HPLC) Rt 11.04 min; m/z 397 [M+H] 98.71%purity.

Example 344-(2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)ethyl)morpholine(Compound 28)

Compound 28 was synthesised following similar procedures outlined inExample 31 (Compound 18), using 4-(2-hydrazinylethyl)morpholine insteadof ethyl hydrazine in Step 3. As a slightly modified procedure, Step 3was performed at 60° C. for 16 h. The reaction mixture was concentratedin vacuo and the crude residue was purified by column chromatography(silica gel, gradient 50 to 100% ethyl acetate/isohexane), followed bypreparative HPLC, yielding Compound 28 as a solid.

LCMS (10 cm_ESCI_Formic) Rt 2.85 min; m/z420 [M+H] 97.41% purity.

Example 354-(4-chloro-1-methyl-5-phenyl-1H-pyrazolo[3,4-c]pyridazin-3-yl)benzonitrile(Compound 24)

A degassed mixture of4-chloro-3-(4-iodophenyl)-1-methyl-5-phenyl-1H-pyrazolo[3,4-c]pyridazine(Compound 43, Example 23) (70 mg, 0.16 mmol), Zn(CN)₂ (18 mg, 0.094mmol) and Pd(PPh₃)₄ (18 mg, 0.016 mmol) in DMF (1 mL) was heated at 100°C. for 40 min in a microwave. The reaction mixture was partitionedbetween ethyl acetate and water. The organic phase was washed with waterand brine, dried using a phase separating cartridge and concentrated invacuo. The crude residue was purified by column chromatography (silicagel, 10 to 20% ethyl acetate/isohexane), yielding Compound 24 as a solid(30 mg).

LCMS (10 cm_ESCI_Formic) Rt 4.4 min; m/z 346 [M+H] 96.24% purity.

Example 363-(4-chloro-1-methyl-5-phenyl-1H-pyrazolo[3,4-c]pyridazin-3-yl)benzonitrile(Compound 34)

Compound 34 was synthesised following similar procedures outlined inExample 35 (Compound 24), using4-chloro-3-(3-iodophenyl)-1-methyl-5-phenyl-1H-pyrazolo[3,4-c]pyridazine(Compound 3, Example 30) as the starting material instead of4-chloro-3-(4-iodophenyl)-1-methyl-5-phenyl-1H-pyrazolo[3,4-c]pyridazine(Compound 43, Example 23).

LCMS (10 cm_ESCI_Formic) Rt 4.36 min; m/z 346 [M+H] 99.13% purity.

Example 373-(3-(4-chloro-1-methyl-5-phenyl-1H-pyrazolo[3,4-c]pyridazin-3-yl)phenyl)prop-2-yn-1-ol(Compound 30)

A mixture of4-chloro-3-(3-iodophenyl)-1-methyl-5-phenyl-1H-pyrazolo[3,4-c]pyridazine(Compound 7, Example 32) (80 mg, 0.18 mmol), propargyl alcohol (41 mg,0.74 mmol), triethylamine (87 μl, 0.63 mmol), CuI (3.5 mg, 0.018 mmol),triphenylphosphine (5 mg, 0.018 mmol) and Pd(PPh₃)₄ (20 mg, 0.018 mmol)in 1,4-dioxane (1.5 mL) was heated at 100° C. for 4 h in a sealed tube.The reaction mixture was partitioned between CH₂Cl₂ and water. Theorganic phase was separated and dried using a phase separating cartridgethen concentrated in vacuo. The crude residue was purified by columnchromatography (silica gel, 20-30% ethyl acetate/isohexane), yieldingCompound 30 as a solid (26 mg).

LCMS (15 cm_Formic_ASCENTIS_HPLC) Rt 9.67 min; m/z 375 [M+H] 86.09%purity.

Example 38N-(3-(2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)acetamido)propyl)-5-((4S)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide(Compound 44)

To a solution ofN-(3-aminopropyl)-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide(also known as N-(3-Aminopropyl)biotinamide trifluoroacetate,commercially available from Sigma-Aldrich or J&K Scientific Ltd) (0.22mmol) in anhydrous DMF (1.1 mL) were sequentially added DIPEA (57 μL,0.33 mmol),2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)acetic acid(80 mg, 0.22 mmol) and2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU, commercially available from Sigma-Aldrich orNovabiochem) (125 mg, 0.33 mmol). The reaction mixture was stirred atroom temperature for 2 h, then diluted with ethyl acetate and washedwith aqueous NaHCO₃ and 4% aqueous LiCl. The organic phase was thendried over MgSO₄, and evaporated under vacuum. The residue was purifiedby preparative HPLC yielding Compound 44 as a beige solid (34 mg).

LCMS (15 cm_Bicarb_GeminiNX_HPLC) Rt 9.26 min; m/z 647 [M+H] 90.15%purity.

Example 392-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)-1-(pyrrolidin-1-yl)ethanone(Compound 45)

To a suspension of2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)acetic acid(510 mg, 1.4 mmol) in CH₂Cl₂ (9 mL) were added pyrrolidine (174 μL, 2.1mmol), diisopropylethylamine (DIPEA) (366 μL, 2.1 mmol) and2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU) (798 mg, 2.1 mmol). The solution obtained wasstirred at room temperature for 90 min then diluted with CH₂Cl₂. Theorganic phase was washed with aqueous NaHCO₃, passed over a phaseseparator and concentrated in vacuo. The resultant residue was purifiedusing chromatography (silica gel, gradient 0 to 70% ethylacetate/isohexane) followed by a second chromatography purification(silica gel, gradient 0 to 25% dichloromethane/ether) yielding Compound45 as a solid (165 mg).

LCMS (10 cm_ESCI_Formic) Rt 4.27 min; m/z 418 [M+H] 99.92% purity.

Example 402-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)acetic acid(Compound

A suspension of sodium2-(5-amino-3-phenyl-4-(phenylethynyl)-1H-pyrazol-1-yl)acetate (2 g, 6.0mmol) in diethylether (10 mL) and concentrated HCl (50 mL) was cooled at−15° C. before sodium nitrite (0.62 g, 8.0 mmol) in water (5 mL) wasadded dropwise. The solution was allowed to warm up to room temperatureand left stirring for further 18 h. The reaction mixture was carefullyquenched with an aqueous solution of Na₂CO₃ at 10° C., until pH basic,and then extracted EtOAc, backwashed with water then brine, dried(MgSO₄) and concentrated in vacuo. The resultant residue was purifiedusing chromatography (silica gel, gradient 2 to 10% diethylether/CH₂Cl₂)yielding Compound 47 as a yellow foam (580 mg).

LCMS (10 cm_ESCI_Formic) Rt 3.73 min; m/z 365 [M+H] 94.82% purity

Example 412-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)-1-morpholinoethanone(Compound 48)

To a suspension of2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)acetic acid(60 mg, 0.16 mmol) in DMF (1 mL) were added morpholine (17 mg, 0.20mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride(EDC. HCl) (38 mg, 0.20 mmol). The solution obtained was stirred at roomtemperature for 18 h then diluted with EtOAc. The organic phase waswashed with water and brine, dried (MgSO₄) and concentrated in vacuo.The resultant residue was purified using chromatography (silica gel,ethyl acetate) yielding the Compound 48 as a solid (20 mg).

LCMS (10 cm_ESCI_Formic) Rt 3.67 min; m/z 434 [M+H] 95.17% purity.

Example 422-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)-N-(cyanomethyl)-N-methylacetamide(Compound 49)

To a suspension of2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)acetic acid(60 mg, 0.16 mmol) in DMF (1 mL) were added N-methyl aminoacetonitrile(14 mg, 0.20 mmol) and 1,1′-carbonyldiimidazole hydrochloride (CDI. HCl)(38 mg, 0.20 mmol). The solution obtained was stirred at roomtemperature for 2 h then diluted with EtOAc. The organic phase waswashed with water and brine, dried (MgSO₄) and concentrated in vacuo.The resultant residue was purified using chromatography (silica gel,gradient 1/1 ethyl acetate/isohexane) yielding Compound 49 as a solid(30 mg).

LCMS (10 cm_ESCI_Formic) Rt 4.17 min; m/z 417 [M+H] 94.82% purity.

Example 432-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)-N-(2-morpholinoethyl)acetamide(Compound 50)

To a suspension of2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)acetic acid(60 mg, 0.16 mmol) in DMF (1 mL) were added 4-(2-aminoethyl)morpholine(32 mg, 0.25 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (EDC. HCl) (38 mg, 0.20 mmol). The solution obtained wasstirred at room temperature for 18 h then diluted with EtOAc. Theorganic phase was washed with water and brine, dried (MgSO₄) andconcentrated in vacuo. The resultant residue was purified usingchromatography (silica gel, gradient 10% MeOH/ethyl acetate) yieldingCompound 50 as a solid (10 mg).

LCMS (10 cm_ESCI_Formic) Rt 3.64 min; m/z 477 [M+H] 95.28% purity.

Example 442-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)-N-methyl-N-(1-methylpiperidin-4-yl)acetamide(Compound 51)

To a suspension of2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)acetic acid(60 mg, 0.16 mmol) in DMF (1 mL) were added1-methyl-4-(methylamino)piperidine (23 mg, 0.18 mmol),diisopropylethylamine (DIPEA) (63 μL, 0.36 mmol) and2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU) (75 mg, 0.20 mmol). The solution obtained wasstirred at room temperature for 1 hr then diluted with EtOAc. Theorganic phase was washed with water and brine, dried (MgSO₄) andconcentrated in vacuo. The resultant residue was purified usingchromatography (silica gel, gradient 1% Et₃N in 10-20% MeOH/ethylacetate) yielding Compound 51 as a solid (2 mg).

LCMS (15 cm_Formic_ASCENTIS_HPLC) Rt 7.79 min; m/z 475 [M+H] 94.53%purity.

Example 452-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)-1-(4-(methylsulfonyl)piperazin-1-yl)ethanone(Compound 52)

To a suspension of2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)acetic acid(56 mg, 0.15 mmol) in DMF (1 mL) were added 1-(methylsulfonyl)piperazine(28 mg, 0.17 mmol), diisopropylethylamine (DIPEA) (59 μL, 0.34 mmol) and2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU) (70 mg, 0.18 mmol). The solution obtained wasstirred at room temperature for 18 h then diluted with EtOAc. Theorganic phase was washed with water and brine, dried (MgSO₄) andconcentrated in vacuo. The resultant residue was purified usingchromatography (silica gel, gradient 20-40% ethyl acetate/isohexane)followed by a second chromatography purification (silica gel, gradient10% ether/CH₂Cl₂) yielding Compound 52 as a solid (11 mg).

LCMS (15 cm_Bicarb_GeminiNX_HPLC) Rt 10.35 min; m/z 511 [M+H] 92.47%purity.

Example 462-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)-N-(2-(dimethylamino)ethyl)-N-methylacetamide(Compound 53)

To a suspension of2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)acetic acid(56 mg, 0.15 mmol) in DMF (1 mL) were addedN¹,N¹,N²-trimethylethane-1,2-diamine (17 mg, 0.17 mmol),diisopropylethylamine (DIPEA) (59 μL, 0.34 mmol) and2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU) (70 mg, 0.18 mmol). The solution obtained wasstirred at room temperature for 18 h then diluted with EtOAc. Theorganic phase was washed with water and brine, dried (MgSO₄) andconcentrated in vacuo. The resultant residue was purified by preparativeHPLC yielding Compound 53 as a solid (11 mg).

LCMS (10 cm_ESCI_Bicarb) Rt 3.35 min; m/z 449 [M+H] 95.56% purity.

Example 472-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)-1,1-dioxido-4-thiomorpholinoethanone(Compound 54)

To a suspension of2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)acetic acid(100 mg, 0.27 mmol) in DMF (2 mL) were added thiomorpholine 1,1-dioxide(40 mg, 0.30 mmol), diisopropylethylamine (DIPEA) (105 μL, 0.60 mmol)and 2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU) (125 mg, 0.33 mmol). The solution obtainedwas stirred at room temperature for 18 h then diluted with EtOAc. Theorganic phase was washed with water and brine, dried (MgSO₄) andconcentrated in vacuo. The resultant residue was purified by preparativeHPLC followed by a second chromatography purification (silica gel,gradient 1/1 ethyl acetate/isohexane) yielding Compound 54 as a solid(33 mg).

LCMS (10 cm_Formic_ACE 3 C18 AR_HPLC) Rt 11.54 min; m/z 482 [M+H] 90.61%purity.

Example 482-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)-1-(4-hydroxypiperidin-1-yl)ethanone(Compound 55)

To a suspension of2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)acetic acid(100 mg, 0.27 mmol) in DMF (2 mL) were added piperidin-4-ol (30 mg, 0.30mmol), diisopropylethylamine (DIPEA) (105 μL, 0.60 mmol) and2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU) (125 mg, 0.33 mmol). The solution obtainedwas stirred at room temperature for 18 h then diluted with EtOAc. Theorganic phase was washed with water and brine, dried (MgSO₄) andconcentrated in vacuo. The resultant residue was purified by preparativeHPLC followed by a second chromatography purification (silica gel,gradient 1/1 ethyl acetate/isohexane) yielding Compound 55 as a solid(10 mg).

LCMS (15 cm_Bicarb_GeminiNX_HPLC) Rt 9.83 min; m/z 448 [M+H] 95.96%purity.

Example 492-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)-1-(4-(2,2,2-trifluoroethyl)piperazin-1-yl)ethanone(Compound 56)

To a suspension of2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)acetic acid(100 mg, 0.27 mmol) in DMF (2 mL) were added1-(2,2,2-trifluoroethyl)piperazine (50 mg, 0.30 mmol),diisopropylethylamine (DIPEA) (105 μL, 0.60 mmol) and2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU) (125 mg, 0.33 mmol). The solution obtainedwas stirred at room temperature for 18 h then diluted with EtOAc. Theorganic phase was washed with water and brine, dried (MgSO₄) andconcentrated in vacuo. The resultant residue was purified by preparativeHPLC yielding Compound 56 as a solid (41 mg).

LCMS (15 cm_Bicarb_GeminiNX_HPLC) Rt 10.93 min; m/z 515 [M+H] 93.62%purity.

Example 502-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)-N-(pyridin-3-ylmethyl)acetamide(Compound 57)

To a suspension of2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)acetic acid(100 mg, 0.27 mmol) in DMF (2 mL) were added pyridin-3-ylmethanamine (33mg, 0.30 mmol), diisopropylethylamine (DIPEA) (105 μL, 0.60 mmol) and2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU) (125 mg, 0.33 mmol). The solution obtainedwas stirred at room temperature for 18 h then diluted with EtOAc. Theorganic phase was washed with water and brine, dried (MgSO₄) andconcentrated in vacuo. The resultant residue was purified by preparativeHPLC yielding Compound 57 as a solid (37 mg).

LCMS (10 cm_ESCI_Formic) Rt 3.24 min; m/z 455 [M+H] 99.50% purity.

Example 511-(2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)acetoyl)piperidine-3-carbonitrile(Compound 58)

To a suspension of2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)acetic acid(100 mg, 0.27 mmol) in DMF (2 mL) were added piperidine-3-carbonitrile(33 mg, 0.30 mmol), diisopropylethylamine (DIPEA) (105 μL, 0.60 mmol)and 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU) (125 mg, 0.33 mmol). The solution obtainedwas stirred at room temperature for 18 h then diluted with EtOAc. Theorganic phase was washed with water and brine, dried (MgSO₄) andconcentrated in vacuo. The resultant residue was purified by preparativeHPLC followed by a second chromatography purification (silica gel,gradient 1/1 ethyl acetate/isohexane) yielding Compound 58 as a solid(28 mg).

LCMS (10 cm_Formic_ACE 3 C18 AR_HPLC) Rt 12.04 min; m/z 457 [M+H] 97.93%purity.

Example 522-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)-1-(4-(dimethylamino)piperidin-1-yl)ethanone(Compound 59)

To a suspension of2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)acetic acid(100 mg, 0.27 mmol) in DMF (2 mL) were addedN,N-dimethylpiperidin-4-amine (40 mg, 0.30 mmol), diisopropylethylamine(DIPEA) (105 μL, 0.60 mmol) and2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU) (125 mg, 0.33 mmol). The solution obtainedwas stirred at room temperature for 18 h then diluted with EtOAc. Theorganic phase was washed with water and brine, dried (MgSO₄) andconcentrated in vacuo. The resultant residue was purified by preparativeHPLC yielding Compound 59 as a solid (10 mg).

LCMS (15 cm_Formic_ASCENTIS_HPLC) Rt 7.99 min; m/z 475 [M+H] 91.14%purity.

Example 532-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)-N-(1-methylpiperidin-4-yl)acetamide(Compound 60)

To a suspension of2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)acetic acid(100 mg, 0.27 mmol) in DMF (2 mL) were added 1-methylpiperidin-4-amine(35 mg, 0.30 mmol), diisopropylethylamine (DIPEA) (105 μL, 0.60 mmol)and 2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU) (125 mg, 0.33 mmol). The solution obtainedwas stirred at room temperature for 18 h then diluted with EtOAc. Theorganic phase was washed with water and brine, dried (MgSO₄) andconcentrated in vacuo. The resultant residue was purified by preparativeHPLC yielding Compound 60 as a solid (22 mg).

LCMS (15 cm_Formic_ASCENTIS_HPLC) Rt 7.90 min; m/z 461 [M+H] 94.77%purity.

Example 542-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)-N-(1-methylcyclobutyl)acetamide(Compound 61)

To a suspension of2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)acetic acid(100 mg, 0.27 mmol) in DMF (2 mL) were added 1-methyl-cyclobutyl aminehydrochloride (37 mg, 0.30 mmol), diisopropylethylamine (DIPEA) (150 μL,0.60 mmol) and2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU) (125 mg, 0.33 mmol). The solution obtainedwas stirred at room temperature for 2 h then diluted with EtOAc. Theorganic phase was washed with water and brine, dried (MgSO₄) andconcentrated in vacuo. The resultant residue was purified bychromatography purification (silica gel, gradient 10-20% ethylacetate/isohexane) yielding Compound 61 as a solid (22 mg).

LCMS (15 cm_Bicarb_GeminiNX_HPLC) Rt 10.96 min; m/z 432 [M+H] 96.73%purity.

Example 552-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)-1-(3,3-difluoropyrrolidin-1-yl)ethanone(Compound 62)

To a suspension of2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)acetic acid(100 mg, 0.27 mmol) in DMF (2 mL) were added 3,3-difluoropyrrolidinehydrochloride (43 mg, 0.30 mmol), diisopropylethylamine (DIPEA) (150 μL,0.60 mmol) and2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU) (125 mg, 0.33 mmol). The solution obtainedwas stirred at room temperature for 2 h then diluted with EtOAc. Theorganic phase was washed with water and brine, dried (MgSO₄) andconcentrated in vacuo. The resultant residue was purified bychromatography purification (silica gel, gradient 10-20% ethylacetate/isohexane) yielding Compound 62 as a solid (18 mg).

LCMS (15 cm_Bicarb_GeminiNX_HPLC) Rt 10.72 min; m/z 454 [M+H] 93.08%purity.

Example 562-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)-1-((1R,4R)-5-methyl-2,5-diazabicyclo[2.2.1]heptan-2-yl)ethanone(Compound 63)

To a suspension of2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)acetic acid(100 mg, 0.27 mmol) in DMF (2 mL) were added(1R,4R)-2-methyl-2,5-diazabicyclo[2.2.1]heptane (26 mg, 0.30 mmol),diisopropylethylamine (DIPEA) (105 μL, 0.60 mmol) and2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU) (125 mg, 0.33 mmol). The solution obtainedwas stirred at room temperature for 18 h then diluted with EtOAc. Theorganic phase was washed with water and brine, dried (MgSO₄) andconcentrated in vacuo. The resultant residue was purified by preparativeHPLC yielding Compound 63 as a solid (11 mg).

LCMS (15 cm_Formic_ASCENTIS_HPLC) Rt 7.82 min; m/z 459 [M+H] 94.39%purity.

Example 572-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)acetamide(Compound 64)

To a solution of2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)acetic acid(160 mg, 0.44 mmol) in CH₂Cl₂ (6 mL) was added oxalyl chloride (55 μl,0.66 mmol). The solution obtained was stirred at room temperature for 1h. The reaction mixture was concentrated in vacuo. The residue wasdissolved in CH₂Cl₂ (6 mL) and a solution (7 N) of ammonia in methanolwas added dropwise (125 μl, 0.8 mmol). The reaction mixture was stirredfor 1 h, then concentrated in vacuo. The resultant residue was purifiedby preparative HPLC yielding Compound 64 as a solid (32 mg).

LCMS (10 cm_ESCI_Bicarb MeCN) Rt 3.62 min; m/z 364 [M+H] 99.1% purity.

Example 582-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)-1-(4-methyl-1,4-diazepan-1-yl)ethanone(Compound 65)

A solution of diisopropylethylamine (DIPEA) (120 μL, 0.67 mmol) and1-methyl 1,4-diazepane (34 mg, 0.3 mmol) in CH₂Cl₂ (1 mL) was added to amixture of2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)acetic acid(100 mg, 0.27 mmol), N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride (EDC) (60 mg, 0.3 mmol) and 2-hydroxypyridine-N-oxide(HOPO) (30 mg, 0.27 mmol). The solution obtained was stirred at roomtemperature for 18 h. The reaction was purified using chromatography(silica gel, gradient 0 to 5% triethylamine/acetone) followed bypreparative HPLC yielding Compound 65 as a white solid (25 mg).

LCMS (10 cm_ESCI_Formic) Rt 2.73 min; m/z 461 [M+H] 99.86% purity.

Example 591-(4-acetoylpiperazin-1-yl)-2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)ethanone(Compound 66)

A solution of diisopropylethylamine (DIPEA) (120 μL, 0.67 mmol) and1-acetylpiperazine (38 mg, 0.3 mmol) in CH₂Cl₂ (1 mL) was added to amixture of2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)acetic acid(100 mg, 0.27 mmol), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride (EDC) (60 mg, 0.3 mmol) and 2-hydroxypyridine-N-oxide(HOPO) (30 mg, 0.27 mmol). The solution obtained was stirred at roomtemperature for 18 h then 1-acetylpiperazine (10 mg, 0.08 mmol) andN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) (30mg, 0.15 mmol) were added. After a further 3.5 h the reaction waspurified using chromatography (silica gel, gradient 0 to 5%triethylamine/acetone) followed by preparative HPLC yielding Compound 66as a white solid (32.2 mg).

LCMS (10 cm_ESCI_Formic) Rt 3.67 min; m/z 475 [M+H] 99.58% purity.

Example 602-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)-1-(3-(dimethylamino)pyrrolidin-1-yl)ethanone(Compound 67)

A solution of diisopropylethylamine (DIPEA) (120 μL, 0.67 mmol) andN,N-dimethylpyrrolidine (34 mg, 0.3 mmol) in CH₂Cl₂ (1 mL) was added toa mixture of2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)acetic acid(100 mg, 0.27 mmol), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride (EDC) (60 mg, 0.3 mmol) and 2-hydroxypyridine-N-oxide(HOPO) (30 mg, 0.27 mmol). The solution obtained was stirred at roomtemperature for 18 h then N,N-dimethylpyrrolidine (10 mg, 0.09 mmol) andN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) (30mg, 0.15 mmol) were added. After a further 3.5 h the reaction waspurified using chromatography (silica gel, gradient 0 to 5%triethylamine/acetone) followed by preparative HPLC yielding Compound 67as a white solid (27.3 mg).

LCMS (10 cm_ESCI_Formic) Rt 2.73 min; m/z 461 [M+H] 99.54% purity.

Example 612-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)-N-(3-(dimethylamino)propyl)acetamide(Compound 68)

Example 61 was synthesised following a similar procedure outlined inExample 58, using 3-dimethylaminopropylamine instead of 1-methyl1,4-diazepane. Compound 68 was obtained as a colourless solid (16 mg).

LCMS (10 cm_ESCI_Formic) Rt 2.68 min; m/z 449 [M+H] 97.9% purity.

Example 624-(2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)acetoyl)piperazin-2-one(Compound 69)

To a suspension of2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)acetic acid(111 mg, 0.3 mmol) in CH₂Cl₂ mL) were added 2-oxopiperazine (33 mg, 0.33mmol), diisopropylethylamine (DIPEA) (80 μL, 0.45 mmol) and2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU) (130 mg, 0.33 mmol). The solution obtainedwas stirred at room temperature for 2 h then diluted with CH₂Cl₂. Theorganic phase was washed with aqueous water, dried over MgSO₄, filteredand concentrated in vacuo. The resultant residue was purified usingchromatography (silica gel, gradient 20 to 100% ethyl acetate/isohexane,then 0 to 100% ethyl acetate/acetone) followed by preparative HPLCyielding Compound 69 as a brown solid (69.7 mg).

LCMS (10 cm_ESCI_Bicarb) Rt 3.52 min; m/z 447 [M+H] 98.17% purity.

Example 632-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)-1-(3-hydroxypyrrolidin-1-yl)ethanone(Compound 70)

Example 63 was synthesised following a similar procedure outlined inExample 62, using 3-hydroxypyrrolidine instead of 2-oxopiperazine.Compound 70 was obtained as a white solid (46 mg).

LCMS (10 cm_ESCI_Formic) Rt 3.73 min; m/z 434 [M+H] 99.79% purity.

Example 642-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)-N-(2-hydroxyethyl)acetamide(Compound 71)

Example 64 was synthesised following a similar procedure outlined inExample 62, using ethanolamine instead of 2-oxopiperazine. Compound 71was obtained as a white solid (13 mg).

LCMS (10 cm_ESCI_Bicarb_MeCN) Rt 3.50 min; m/z 408 [M+H] 99.78% purity.

Example 65(S)-2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)-N-(2-hydroxypropyl)acetamide(Compound 72)

Example 65 was synthesised following a similar procedure outlined inExample 62, using (S)-1-amino-2-propanol instead of 2-oxopiperazine.Compound 72 was obtained as a white solid (29 mg).

LCMS (10 cm_ESCI_Bicarb_MeCN) Rt 3.58 min; m/z 422 [M+H] 98.12% purity.

Example 662-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)-N-(2-methoxyethyl)acetamide(Compound 73)

Example 66 was synthesised following a similar procedure outlined inExample 62, using methoxyethylamine instead of 2-oxopiperazine. Compound73 was obtained as a white solid (25.7 mg).

LCMS (10 cm_ESCI_Formic_MeCN) Rt 4.05 min; m/z 422 [M+H] 99.65% purity.

Example 67(R)-2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)-N-(2-hydroxypropyl)acetamide(Compound 74)

Example 67 was synthesised following a similar procedure outlined inExample 62, using (R)-1-amino-2-propanol instead of 2-oxopiperazine.Compound 74 was obtained as a white solid (37 mg).

LCMS (10 cm_ESCI_Bicarb_MeCN) Rt 3.14 min; m/z 422 [M+H] 99.7% purity.

Example 682-(4-chloro-3-(4-fluorophenyl)-5-phenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)-1-morpholinoethanone(Compound 75)

Step 1: ethyl 2-(5-amino-3-(4-fluorophenyl)-1H-pyrazol-1-yl)acetate

A mixture of 4-fluorobenzoylacetonitrile (5 g, 30.7 mmol) and ethylhydrazinoacetate hydrochloride (4.74 g, 30.7 mmol) in ethanol (50 mL)was heated to reflux for 2 h. The reaction mixture was concentrated invacuo. The crude reaction mixture was partitioned between CH₂Cl₂ andconcentrated aqueous ammonia. The aqueous phase was extracted withCH₂Cl₂ and the organic phases combined, dried over MgSO₄, filtered andevaporated to give ethyl2-(5-amino-3-(4-fluorophenyl)-1H-pyrazol-1-yl)acetate as a solid (7.4g), which was used as such in the subsequent step.

Step 2: ethyl 2-(5-acetamido-3-(4-fluorophenyl)-1H-pyrazol-1-yl)acetate

To a solution of ethyl2-(5-amino-3-(4-fluorophenyl)-1H-pyrazol-1-yl)acetate (6.91 g, 26.3mmol) in pyridine (62 mL) was added acetic anhydride (3 ml, 31.5 mmol).The reaction was stirred at room temperature for 16 h. Ethanol was addedand the mixture was concentrated in vacuo. The crude residue waspurified by column chromatography (silica gel, gradient 10 to 100% ethylacetate/isohexane) yielding ethyl2-(5-acetamido-3-(4-fluorophenyl)-1H-pyrazol-1-yl)acetate as a solid(7.6 g).

Step 3: ethyl2-(5-acetamido-3-(4-fluorophenyl)-4-iodo-1H-pyrazol-1-yl)acetate

To a solution of ethyl2-(5-acetamido-3-(4-fluorophenyl)-1H-pyrazol-1-yl)acetate (1.1 g, 3.7mmol) in ethanol (33 mL) were added iodic acid (170 mg, 0.95 mmol) andiodine (480 mg, 1.9 mmol). The reaction was stirred for 90 min at 60° C.The reaction mixture was concentrated in vacuo and the residue wasdissolved in CH₂Cl₂ and washed with a 2 N Na₂SO₃ solution. The organicphase was dried over MgSO₄, filtered and evaporated to give ethyl2-(5-acetamido-3-(4-fluorophenyl)-4-iodo-1H-pyrazol-1-yl)acetate as awhite solid (1.63 g), which was used as such in the subsequent step.

Step 4: ethyl2-(5-acetamido-3-(4-fluorophenyl)-4-(phenylethynyl)-1H-pyrazol-1-yl)acetate

A degassed solution of phenylacetylene (290 mg, 2.8 mmol) and Et₃N (11mL) in DMF (4 mL) was added to ethyl2-(5-acetamido-3-(4-fluorophenyl)-4-(phenylethynyl)-1H-pyrazol-1-yl)acetate(1.0 g, 2.3 mmol), CuI (90 mg, 0.46 mmol) and PdCl₂(PPh₃)₂ (160 mg, 0.23mmol) under an atmosphere of N₂. The reaction was then heated to 85° C.for 5 h. The reaction mixture was concentrated in vacuo and the residuewas purified by column chromatography (silica gel, gradient 10 to 100%ethyl acetate/isohexane) yielding ethyl2-(5-acetamido-3-(4-fluorophenyl)-4-(phenylethynyl)-1H-pyrazol-1-yl)acetateas a tan solid (620 mg).

Step 5:2-(5-amino-3-(4-fluorophenyl)-4-(phenylethynyl)-1H-pyrazol-1-yl)aceticacid

A solution of ethyl2-(5-acetamido-3-(4-fluorophenyl)-4-(phenylethynyl)-1H-pyrazol-1-yl)acetate(620 mg, 1.5 mmol) in ethanol (7 mL) and 25% NaOH (7 mL) was heated to83° C. for 16 h. The resultant solid was filtered, washed with ethanoland water then dried in vacuo to give2-(5-amino-3-(4-fluorophenyl)-4-(phenylethynyl)-1H-pyrazol-1-yl)aceticacid as a solid (521 mg), which was used as such in the subsequent step.

Step 6:2-(4-chloro-3-(4-fluorophenyl)-5-phenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)aceticacid

To a cooled (cooling bath −5° C.) stirred suspension of sodium nitrite(320 mg, 4.65 mmol) in conc. HCl (5 mL) was added2-(5-amino-3-(4-fluorophenyl)-4-(phenylethynyl)-1H-pyrazol-1-yl)aceticacid (521 mg, 1.55 mmol). A further 5 ml of conc. HCl was added and thecooling bath was removed and the reaction mixture was stirred at roomtemperature for 3.5 h. Water and CH₂Cl₂ were added to the reactionmixture. The aqueous phase was extracted with CH₂Cl₂ and the organicphases combined, dried over MgSO₄, filtered and evaporated to give2-(4-chloro-3-(4-fluorophenyl)-5-phenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)aceticacid as a solid (419 mg), which was used as such in the subsequent step.

Step 7:2-(4-chloro-3-(4-fluorophenyl)-5-phenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)-1-morpholinoethanone

To a suspension of2-(4-chloro-3-(4-fluorophenyl)-5-phenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)aceticacid (150 mg, 0.39 mmol) in CH₂Cl₂ (3 mL) were added morpholine (34 mg,0.39 mmol), diisopropylethylamine (DIPEA) (200 μL, 1.2 mmol) and2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU) (180 mg, 0.47 mmol). The solution obtainedwas stirred at room temperature for 75 min before being purified usingchromatography (silica gel, gradient 20 to 100% ethyl acetate/isohexane)followed by preparative HPLC yielding Compound 75 as a clear glass solid(11.5 mg).

LCMS (10 cm_ESCI_Formic MeCN) Rt 4.12 min; m/z 452 [M+H] 99.32% purity.

Example 692-(4-chloro-3-(3-fluorophenyl)-5-phenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)-1-morpholinoethanone(Compound 76)

Example 69 (Compound 76) was synthesised following a similar procedureoutlined in Example 68 (Compound 75), using 3-fluorobenzoylacetonitrileinstead of 4-fluorobenzoylacetonitrile in step 1. Compound 76 wasobtained as a yellow solid (4.8 mg).

LCMS (15 cm_Bicarb_GeminiNX_HPLC_CH3CN) Rt 10.39 min; m/z 452 [M+H]95.01% purity.

Example 702-(4-chloro-3-(4-fluorophenyl)-5-phenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)ethanol(Compound 77)

Compound 77 was synthesised starting from ethyl2-(5-acetamido-3-(4-fluorophenyl)-4-(phenylethynyl)-1H-pyrazol-1-yl)acetate(Example 68, Step 4):

Step 1:N-(3-(4-fluorophenyl)-1-(2-hydroxyethyl)-4-(phenylethynyl)-1H-pyrazol-5-yl)acetamide

To a solution of ethyl2-(5-acetamido-3-(4-fluorophenyl)-4-(phenylethynyl)-1H-pyrazol-1-yl)acetate(1 g, 2.5 mmol) in ethanol (40 mL) and methanol (10 ml) was added sodiumborohydride (466 mg, 12.3 mmol and the reaction stirred at roomtemperature for 16 h. The reaction mixture was concentrated in vacuo andthe residue was dissolved in CH₂Cl₂ and washed with a dilute NH₄Clsolution. The aqueous was extracted with CH₂Cl₂ and the combined organicextracts were dried over MgSO₄, filtered and evaporated. The crudeproduct was suspended in CH₂Cl₂ (20 ml), heated to reflux, allowed tocool and the product was filtered and dried in vacuo to giveN-(3-(4-fluorophenyl)-1-(2-hydroxyethyl)-4-(phenylethynyl)-1H-pyrazol-5-yl)acetamideas a white solid (659 mg), which was used as such in the subsequentstep.

Step 2:2-(5-amino-3-(4-fluorophenyl)-4-(phenylethynyl)-1H-pyrazol-1-yl)ethanol

A solution ofN-(3-(4-fluorophenyl)-1-(2-hydroxyethyl)-4-(phenylethynyl)-1H-pyrazol-5-yl)acetamide(382 mg, 1.05 mmol) in ethanol (4 mL) and 25% NaOH (6 mL) was heated to83° C. for 16 h. The reaction mixture was partitioned between ethylacetate and water. The aqueous phase was washed with ethyl acetate twiceand the organic phases combined, dried over MgSO₄, filtered andevaporated, purification using chromatography (silica gel, gradient 0 to10% methanol/CH₂Cl₂) gave2-(5-amino-3-(4-fluorophenyl)-4-(phenylethynyl)-1H-pyrazol-1-yl)ethanolas a solid (122 mg), which was used as such in the subsequent step.

Step 3:2-(4-chloro-3-(4-fluorophenyl)-5-phenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)ethanol

To a cooled (cooling bath −10° C.) stirred suspension of sodium nitrite(120 mg, 1.7 mmol) in conc. HCl (3.6 mL) was added2-(5-amino-3-(4-fluorophenyl)-4-(phenylethynyl)-1H-pyrazol-1-yl)ethanol(183 mg, 0.57 mmol) and the reaction mixture was allowed to warm toambient temperature over 90 min. Water and CH₂Cl₂ were added to thereaction mixture. The aqueous phase was extracted with CH₂Cl₂ (×4) andthe organic phases combined, dried over MgSO₄, filtered and evaporatedthe crude material was purified using chromatography (silica gel,gradient 20 to 100% ethyl acetate/isohexane) followed by preparativeHPLC yielding Compound 77 as a white solid (6.5 mg).

LCMS (10 cm_ESCI_Formic MeCN) Rt 4.07 min; m/z 369 [M+H] 99.76% purity.

Example 714-(2-(4-chloro-3-(4-fluorophenyl)-5-phenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)ethyl)morpholine(Compound 78)

Example 71 (Compound 78) was synthesised starting fromN-(3-(4-fluorophenyl)-1-(2-hydroxyethyl-4-(phenylethynyl)-1H-pyrazol-5-yl)acetamide(Example 70 Step 1) (Compound 77)

Step 1:N-(3-(4-fluorophenyl)-1-(2-morpholinoethyl)-4-(phenylethynyl)-1H-pyrazol-5-yl)acetamide

To a suspension ofN-(3-(4-fluorophenyl)-1-(2-hydroxyethyl)-4-(phenylethynyl)-1H-pyrazol-5-yl)acetamide(269 mg, 0.74 mmol) in CH₂Cl₂ (9 mL) and triethylamine (155 μl, 1.11mmol) was added methanesulphonyl chloride (65 μl, 0.81 mmol and thereaction stirred at room temperature for 16 h. The reaction mixture waspoured onto ice/water and the organic layer was dried over MgSO₄,filtered and evaporated. The crude product was dissolved in THF (10 ml)and morpholine (320 μl, 3.7 mmol) was added. The reaction was heated to55° C. for 24 h before evaporating the solvent in vacuo. The crude waspartitioned between CH₂Cl₂ and dilute aqueous NaHCO₃ and the organiclayer was dried over MgSO₄, filtered and evaporated. Purification usingchromatography (silica gel, gradient 0 to 10% methanol/CH₂Cl₂) gaveN-(3-(4-fluorophenyl)-1-(2-morpholinoethyl)-4-(phenylethynyl)-1H-pyrazol-5-yl)acetamideas a white solid (141 mg) which was used as such in the subsequent step.

Step 2:3-(4-fluorophenyl)-1-(2-morpholinoethyl)-4-(phenylethynyl)-1H-pyrazol-5-amine

A solution ofN-(3-(4-fluorophenyl)-1-(2-morpholinoethyl)-4-(phenylethynyl)-1H-pyrazol-5-yl)acetamide(141 mg, 0.32 mmol) in ethanol (1.5 mL) and 25% NaOH (1.5 mL) was heatedto 83° C. for 16 h. The reaction mixture was partitioned between ethylacetate and water. The aqueous phase was washed with ethyl acetate andthe organic phases combined, dried over MgSO₄, filtered and evaporatedto give3-(4-fluorophenyl)-1-(2-morpholinoethyl)-4-(phenylethynyl)-1H-pyrazol-5-amineas a solid (129 mg) which was used as such in the subsequent step.

Step 3:3-(4-fluorophenyl)-1-(2-morpholinoethyl)-5-phenyl-1H-pyrazolo[3,4-c]pyridazin-4-ol

To a cooled (cooling bath −10° C.) stirred suspension of sodium nitrite(82 mg, 1.2 mmol) in conc. HCl (2.6 mL) was added a solution of3-(4-fluorophenyl)-1-(2-morpholinoethyl)-4-(phenylethynyl)-1H-pyrazol-5-amine(129 mg, 0.33 mmol) in trifluoroacetic acid (0.5 ml) and the reactionmixture was allowed to warm to ambient temperature over 2 h. Water andCH₂Cl₂ were added. The aqueous phase was extracted with CH₂Cl₂ (×3) andthe organic phases combined, dried over MgSO₄, filtered and evaporated.The crude material was purified by preparative HPLC to give3-(4-fluorophenyl)-1-(2-morpholinoethyl)-5-phenyl-1H-pyrazolo[3,4-c]pyridazin-4-olas a solid (27.8 mg) which was used as such in the subsequent step.

Step 4:4-(2-(4-chloro-3-(4-fluorophenyl)-5-phenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)ethyl)morpholine(Compound 78)

3-(4-fluorophenyl)-1-(2-morpholinoethyl)-5-phenyl-1H-pyrazolo[3,4-c]pyridazin-4-ol(27.8 mg, 0.07 mmol) was heated to 80° C. in POCl₃ (0.5 ml) for 2.75 hthe solvent was removed in vacuo and the crude purified by preparativeHPLC to give Compound 78 as an orange solid (10.7 mg).

LCMS (10 cm_ESCI_Formic_MeCN) Rt 2.87 min; m/z 438 [M+H] 99.73% purity.

Example 722-(4-chloro-3-(3-fluorophenyl)-5-phenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)-1-(pyrrolidin-1-yl)ethanone(Compound 79)

Example 72 (Compound 79) was synthesised following a similar procedureoutlined in Example 68 (Compound 75), using 3-fluorobenzoylacetonitrileinstead of 4-fluorobenzoylacetonitrile in step 1 and pyrrolidine insteadof morpholine in step 7. Compound 79 was obtained as a yellow solid(13.1 mg).

LCMS (15 cm_Bicarb_GeminiNX_HPLC_CH3CN) Rt 10.68 min; m/z 436 [M+H]96.6% purity.

Example 732-(4-chloro-3-(4-fluorophenyl)-5-phenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)-1-(pyrrolidin-1-yl)ethanone(Compound 80)

Example 73 (Compound 80) was synthesised following a similar procedureoutlined in Example 68, using pyrrolidine instead of morpholine in step7. Compound 80 was obtained as a yellow solid (13.1 mg).

LCMS (15 cm_Bicarb_GeminiNX_HPLC_CH3CN) Rt 10.66 min; m/z 436 [M+H]95.6% purity.

Example 744-chloro-1-(2-(4-methylpiperazin-1-yl)ethyl)-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazine(Compound 81)

Example 74 was synthesised following a similar procedure outlined inExample 71, using N-methyl piperazine instead of morpholine in step 1.Compound 81 was obtained as a yellow solid (6.0 mg).

LCMS (15 cm_Formic_ASCE+NTIS_HPLC_CH3CN) Rt 8.01 min; m/z 433 [M+H]98.4% purity.

Example 75 4-chloro-3,5-diphenyl-1-vinyl-1H-pyrazolo[3,4-c]pyridazine(Compound 82)

Compound 82 was synthesised starting from2-(4-chloro-3,5-diphenyl-1H-pyrazolo-[3,4-c]pyridazin-1-yl)ethanol(Example 32)

Step 1:4-chloro-1-(2-iodoethyl)-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazine

Triphenylphosphine (160 mg, 0.62 mmol), imidazole (42 mg, 0.62 mmol) andiodine (160 mg, 0.62 mmol) were added to a solution of2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)ethanol (181mg, 0.52 mmol) in CH₂Cl₂ (6 ml). After stirring at ambient temperaturefor 1 h the reaction was filtered and solvent removed in vacuo.Purification using chromatography (silica gel, gradient 10 to 60% ethylacetate/isohexane) gave4-chloro-1-(2-iodoethyl)-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazine as aclear oil (202 mg) which was used as such in the subsequent step.

Step 2: 4-chloro-3,5-diphenyl-1-vinyl-1H-pyrazolo[3,4-c]pyridazine(Compound 82)

A solution of4-chloro-1-(2-iodoethyl)-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazine (53mg, 0.11 mmol) in CH₂Cl₂ (1.5 ml) was added to trimethylethylene diamine(97 mg, 0.95 mmol) and the mixture stirred at ambient temperature for 3days. The solvent was removed in vacuo and the crude purified bychromatography (silica gel, gradient 5 to 100% ethyl acetate/isohexane)to give Compound 82 as a yellow solid (12 mg).

LCMS (15 cm_Bicarb_GeminiNX_HPLC_CH3CN) Rt 11.63 min; m/z 333 [M+H]95.16% purity.

Example 76(R)-1-(2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)ethyl)pyrrolidin-3-ol(Compound 83)

A solution of (R)-3-pyrrolidinol (166 mg, 1.9 mmol) in CH₂Cl₂ (1.5 ml)was added to4-chloro-1-(2-iodoethyl)-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazine(example 74 step 1) (113 mg, 0.24 mmol) and the reaction stirred atambient for 24 h. The resultant residue was purified usingchromatography (silica gel, gradient 20 to 100% ethyl acetate/isohexane,then 0 to 100% ethyl acetate/acetone) followed by preparative HPLC togive Compound 83 as an orange solid (17 mg).

LCMS (15 cm_Bicarb_GeminiNX_HPLC_CH3CN) Rt 10.32 min; m/z 420 [M+H]97.06% purity.

Example 774-(2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)ethyl)-1-methylpiperazin-2-one(Compound 84)

A solution of4-chloro-1-(2-iodoethyl)-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazine(example 74 step 1 (Compound 81)) (100 mg, 0.21 mmol) in CH₂Cl₂ (1 ml)was added to piperazinone hydrochloride (160 mg, 1.05 mmol) anddiisopropylethylamine (DIPEA) (220 μl, 12.6 mmol) in CH₂Cl₂ (0.5 ml) andthe reaction stirred at ambient for 2 days. Further piperazinonehydrochloride (160 mg, 1.05 mmol) and diisopropylethylamine (DIPEA) (220μl, 12.6 mmol) were added and the reaction stirred for a further 24 h.The resultant residue was purified using chromatography (silica gel,gradient 20 to 100% ethyl acetate/isohexane, then 0 to 100% ethylacetate/acetone) followed by preparative HPLC to give Compound 84 as ayellow glass solid (17 mg).

LCMS (15 cm_Bicarb_GeminiNX_HPLC_CH3CN) Rt 10.21 min; m/z 447 [M+H]97.76% purity.

Example 781-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)-2-methylpropan-2-ol(Compound 85)

Step 1:N-(1-(2-hydroxy-2-methylpropyl)-3-phenyl-4-(phenylethynyl)-1H-pyrazol-5-yl)acetamide

To a solution of ethyl2-(5-acetamido-3-phenyl-4-(phenylethynyl)-1H-pyrazol-1-yl)acetate (1.0g, 2.58 mmol) in tetrahydrofuran (THF, 26 mL) was added methyl magnesiumchloride (3 M solution in THF, 3 mL, 9 mmol) at 0° C. The solutionobtained was stirred at room temperature for 3.5 h then successivelydiluted with ethyl acetate and quenched by addition of 1 M hydrochloricacid. The aqueous phase was extracted with ethyl acetate, and thecombined organic layers were dried over MgSO₄ and concentrated in vacuo.The resultant residue was purified using chromatography (silica gel,gradient 0 to 75% ethyl acetate/isohexane) yieldingN-(1-(2-hydroxy-2-methylpropyl)-3-phenyl-4-(phenylethynyl)-1H-pyrazol-5-yl)acetamideas a solid (529 mg).

Step 2:1-(5-amino-3-phenyl-4-(phenylethynyl)-1H-pyrazol-1-yl)-2-methylpropan-2-ol

1-(5-Amino-3-phenyl-4-(phenylethynyl)-1H-pyrazol-1-yl)-2-methylpropan-2-olwas synthesised following similar procedures outlined in Example 1 usingN-(1-(2-hydroxy-2-methylpropyl)-3-phenyl-4-(phenylethynyl)-1H-pyrazol-5-yl)acetamidein step 5.

Step 3:1-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)-2-methylpropan-2-ol

To cooled (cooling bath −15° C.) conc. HCl (9 mL) was added sodiumnitrite in one portion (121 mg, 1.75 mmol) and the suspension was leftto stir for 10 min after which1-(5-amino-3-phenyl-4-(phenylethynyl)-1H-pyrazol-1-yl)-2-methylpropan-2-ol(290 mg, 0.88 mmol) was added. After 5 min, the cooling bath was removedand the reaction mixture was stirred at room temperature for 3 h. Thereaction was cooled again (0° C.) and DCM was added followed by water.The aqueous phase was extracted with DCM and the organic phases werecombined, dried over MgSO₄, filtered and evaporated. Crude material waspurified by column chromatography (silica gel, gradient 0 to 50% ethylacetate/isohexane) yielding1-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)-2-methylpropan-2-olas an orange oil (56 mg). The material obtained was further purified bypreparative HPLC, yielding 34 mg of Compound 85 as a solid.

LCMS (10 cm_ESCI_Formic_MeCN) Rt 4.49 min; m/z 379 [M+H] 99.71% purity.

Example 79 methyl2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)acetate(Compound 86)

A suspension of2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)acetic acid(528 mg, 1.45 mmol) in thionyl chloride (10 mL) was heated at 70° C. forone hour. The solvent was evaporated under reduced pressure and theresidue was dissolved in anhydrous CH₂Cl₂. Anhydrous methanol (65 μL,1.6 mmol) was added at 0° C. and the reaction mixture was allowed toslowly reach room temperature overnight. After removal of solvents underreduced pressure, the crude mixture was directly loaded onto a silicagel column and the product was eluted using mixtures of ethylacetate/isohexane (gradient 0 to 40%). Half of the material obtained wasfurther purified by preparative HPLC, yielding 13.4 mg of Compound 86.

LCMS (10 cm_ESCI_Bicarb_MeCN) Rt 10.9 min; m/z 379 [M+H] 94.66% purity.

Example 801-(azetidin-1-yl)-2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)ethanone(Compound 87)

1-(Azetidin-1-yl)-2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)ethanonewas synthesised following similar procedures outlined in Example 39using 2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)aceticacid. Chromatography (silica gel, gradient 0 to 70% ethylacetate/isohexane) followed by preparative HPLC purification yielded 17mg of Compound 87.

LCMS (10 cm_ESCI_Formic) Rt 10.34 min; m/z 404 [M+H] 97.37% purity.

Example 81(S)-2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)-1-(3-fluoropyrrolidin-1-yl)ethanone(Compound 88)

A solution of2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)acetic acid(170 mg, 0.47 mmol) in thionyl chloride (4.7 mL) was heated at 60° C.for 1.5 h. The solvent was evaporated under reduced pressure and theresidue was dissolved in anhydrous CH₂Cl₂ (4.7 mL). The reaction mixturewas split into two tubes and (S)-fluoropyrrolidin hydrochloride (125 mg,0.26 mmol), followed by triethylamine (72 μL, 0.52 mmol) was added toone of them. The reaction mixture was stirred at room temperatureovernight, then concentrated under reduced pressure. The resultantresidue was purified using chromatography (silica gel, gradient 0 to 25%diethyl ether/CH₂Cl₂) to yield 15 mg of Compound 88.

LCMS (10 cm_ESCI_Bicarb_MeCN) Rt 4.0 min; m/z 436 [M+H] 96.15% purity.

Example 82(R)-2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)-1-(3-fluoropyrrolidin-1-yl)ethanone(Compound 89)

(R)-2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)-1-(3-fluoropyrrolidin-1-yl)ethanonewas synthesised following similar procedures outlined in Example 81 andpurified by chromatography (silica gel, gradient 0 to 25% diethylether/CH₂Cl₂) to yield 17 mg of Compound 89.

LCMS (10 cm_ESCI_Bicarb_MeCN) Rt 3.97 min; m/z 436 [M+H] 96.09% purity.

Example 832-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)-1-(3,3-difluoroazetidin-1-yl)ethanone(Compound 90)

2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)-1-(3,3-difluoroazetidin-1-yl)ethanonewas synthesised following similar procedures outlined in Example 81 andpurified by preparative HPLC, yielding 29 mg of Compound 90 as a whitesolid.

LCMS (10 cm_ESCI_Bicarb_MeCN) Rt 3.71 min; m/z 440 [M+H] 98.92% purity.

Example 841-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)propan-2-ol(Compound 91)

Step 1:2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)acetaldehyde

To a solution of2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)ethanol (300mg, 0.86 mmol) in CH₂Cl₂ (5.7 mL) was added Dess-Martin periodinane(DMP, 437 mg, 1.03 mmol) at room temperature. The mixture was stirred atroom temperature for 35 min, then the solids were filtered off and thefiltrate was stirred with aqueous NaHCO₃ and Na₂S₂O₃ for one hour. Theaqueous phase was re-extracted with CH₂Cl₂ and the combined organiclayers were passed over a phase separator and concentrated under reducedpressure. The crude aldehyde2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)acetaldehyde(384 mg) was used directly in the following step.

Step 2:1-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)propan-2-ol(Compound 91)

A solution of2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)acetaldehyde(128 mg, 0.29 mmol) in THF (2.9 mL) was added dropwise over 30 min to asolution of methyl magnesium chloride (290 μL of 3 M solution diluted in2.9 mL THF) at 0° C. The crude mixture was partitioned between CH₂Cl₂and 2 M hydrochloric acid. The aqueous phase was extracted with CH₂Cl₂and the combined organic layers were passed over a phase separator andconcentrated under reduced pressure. The reaction was repeated withanother 80 mg of starting2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)acetaldehydeand the combined crude mixtures were purified by column chromatography(silica gel, gradient 50 to 100% ethyl acetate/isohexane). Additionalpreparative HPLC purification yielded 7.2 mg of Compound 91.

LCMS (10 cm_ESCI_Bicarb_MeCN) Rt 10.57 min; m/z 365 [M+H] 96.2% purity.

Example 851-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)propan-2-ol(Compound 92)

Step 1: 4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazine

To a solution of4-chloro-3,5-diphenyl-1-vinyl-1H-pyrazolo[3,4-c]pyridazine (50 mg, 0.15mmol) in acetone (5 mL) was added a 2% aqueous solution of potassiumpermanganate (71 mg, 0.45 mmol, 3.5 mL water) in one portion. Themixture was stirred at room temperature for 2 h, then another portion ofpotassium permanganate (12 mg, 0.075 mmol), was added and stirringcontinued for 15 min. The solids were filtered off and the filtrate waspartitioned between water and CH₂Cl₂. The aqueous phase was re-extractedwith CH₂Cl₂ and the combined organic layers were dried over MgSO₄ andconcentrated under reduced pressure. The crude4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazine (64 mg) was useddirectly in the following step.

Step 2:1-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)propan-2-ol(Compound 92)

A solution of 4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazine (45 mg,0.15 mmol) in dimethylformamide (DMF, 1.5 mL) was successively treatedwith cesium carbonate (64 mg, 0.2 mmol) and 2-methoxyethyl bromide (31.3mg, 0.23 mmol). The crude mixture was heated at 130° C. in the microwavefor 30 min, then filtered off. The filtrate was evaporated down underreduced pressure and purified by preparative HPLC, yielding 27 mg ofCompound 92.

LCMS (10 cm_ESCI_Bicarb_MeCN) Rt 11.16 min; m/z 365 [M+H] 97.33% purity.

Example 864-chloro-1-(oxetan-3-yl)-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazine(Compound 93)

A solution of 4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazine (50 mg,0.16 mmol) in dioxane (0.5 mL) was treated with oxetan-3-ol (24 mg, 0.32mmol), triphenylphosphine (84 mg, 0.32 mmol) and diethylazodicarboxylate (DEAD, 56 mg, 0.32 mmol) and heated in the microwave at85° C. for 30 min. The crude mixture evaporated down under reducedpressure and purified by preparative HPLC, and subsequently by columnchromatography (silica gel, gradient 0 to 15% diethyl ether/CH₂Cl₂) togive 23 mg of Compound 93 as a white solid.

LCMS (10 cm_ESCI_Formic_MeCN) Rt 10.95 min; m/z 363 [M+H] 98.12% purity.

Example 872-(4-chloro-3-(3-fluorophenyl)-5-phenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)ethanol(Compound 94)

Compound 94 was synthesised starting from ethyl2-(5-acetamido-3-(3-fluorophenyl)-4-(phenylethynyl)-1H-pyrazol-1-yl)acetate.Ethyl2-(5-acetamido-3-(3-fluorophenyl)-4-(phenylethynyl)-1H-pyrazol-1-yl)acetatewas synthesised following a similar procedure outlined in Example 68(steps 1 to 4), using 3-fluorobenzoylacetonitrile instead of4-fluorobenzoylacetonitrile in step 1.

Step 1:2-(5-amino-3-(3-fluorophenyl)-4-(phenylethynyl)-1H-pyrazol-1-yl)ethanol

To a solution of ethyl2-(5-acetamido-3-(3-fluorophenyl)-4-(phenylethynyl)-1H-pyrazol-1-yl)acetate(3.25 g, 8.02 mmol) in ethanol (90 mL) was added sodium borohydride (610mg, 16.04 mmol) and the reaction stirred at room temperature for 36 h.Additional sodium borohydride was added (305 mg, 8.02 mmol) and thereaction stirred at room temperature for 36 h. 25% NaOH (0.9 mL) wasadded and the reaction mixture was stirred at 80° C. for 3.5 h. Thereaction mixture was partitioned between ethyl acetate and water. Theaqueous phase was washed with ethyl acetate twice and the organic phasescombined, dried over MgSO₄, filtered and evaporated. The crude materialwas suspended in diethylether (30 mL) and the product was filtered anddried in vacuo to give2-(5-amino-3-(3-fluorophenyl)-4-(phenylethynyl)-1H-pyrazol-1-yl)ethanolas a white solid (2.2 g), which was used as such in the subsequent step.

Step 2:2-(4-chloro-3-(3-fluorophenyl)-5-phenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)ethanol

Sodium nitrite (555 mg, 8.04 mmol) was added portionwise to a cooled(cooling bath −10° C.) stirred solution of conc. HCl (11.7 mL). After 15minutes,2-(5-amino-3-(3-fluorophenyl)-4-(phenylethynyl)-1H-pyrazol-1-yl)ethanol(860 mg, 2.68 mmol) was added portionwise and the reaction mixture wasallowed to warm to room temperature over 90 min. Water and CH₂Cl₂ wereadded to the reaction mixture. The aqueous phase was extracted withCH₂Cl₂ (×4) and the organic phases combined, dried over MgSO₄, filteredand evaporated the crude material was purified using chromatography(silica gel, gradient 0 to 50% ethyl acetate/isohexane) followed bypreparative HPLC yielding Compound 94 as a white solid (40 mg).

LCMS (15 cm_Bicarb_GeminiNX_HPLC_CH3CN) Rt 10.39 min; m/z 369 [M+H]96.26% purity.

Example 882-(4-chloro-3-(3,5-difluorophenyl)-5-phenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)ethanol(Compound 95)

Compound 95 was synthesised following a similar procedure outlined inExample 70, using 3-(3,5-difluorophenyl)-3-oxopropanenitrile instead of4-fluorobenzoylacetonitrile in step 1.

Synthesis of 3-(3,5-difluorophenyl)-3-oxopropanenitrile

Diisopropylamine (10.6 mL, 76 mmol) was dissolved in dry THF (10 mL) andthe solution cooled to −20° C. A solution of n-butyllithium (2.5N inhexanes, 30.5 mL, 76 mmol) was added slowly at such a rate as to keepthe internal temperature below 0° C. After stirring for 5 min, thesolution was cooled to −20° C. and added to a solution of acetonitrile(2.37 mL, 45.3 mmol) and ethyl 3,5-difluorobenzoate (6.23 g, 36.2 mmol)in dry THF (18 mL) at such a rate as to keep the temperature below −40°C. A further 10 mL of dry THF was used to transfer the remaining lithiumdiisopropylamide to the reaction. The reaction was allowed to warm toambient over 2 h before being quenched with saturated aqueous ammoniumchloride solution. The reaction was extracted with ethyl acetate (×2)and the extracts discarded. The aqueous was acidified with 2N HCl andextracted with ethyl acetate. The organic extract was washed with 2N HCland brine before being dried over MgSO₄, filtered and evaporated.Purification using chromatography (silica gel, gradient 5 to 100% ethylacetate/isohexane) gave 3-(3,5-difluorophenyl)-3-oxopropanenitrile as ayellow solid (2.82 g).

Compound 95 was synthesised following a similar procedure outlined inExample 70, using 3-(3,5-difluorophenyl)-3-oxopropanenitrile instead of4-fluorobenzoylacetonitrile in step 1. Compound 95 was obtained as a tansolid (30.6 mg).

LCMS (10 cm_Formic_ACE 3 C18 AR_HPLC_CH3CN) Rt 11.89 min; m/z 387 [M+H]95.85% purity.

Example 892-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)propan-1-ol(Compound 96)

Step 1: Ethyl2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)propanoate

Ethyl2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)propanoate wassynthesised following a similar procedure outlined in Example 85, usingethyl 2-bromopropionate instead of 2-methoxyethyl bromide in step 2.

Step 2:2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)propan-1-ol(Compound 96)

To a cooled (0° C.) stirred solution of ethyl2-(4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazin-1-yl)propanoate (72mg, 0.177 mmol) in THF (0.5 mL) was added dropwise a 1.1 M solution ofdiisobutylaluminum hydride in cyclohexane (1 mL, 1.1 mmol). After 15minutes, a saturated solution of ammonium chloride and CH₂Cl₂ were addedat 0° C. to the reaction mixture. The aqueous phase was extracted withCH₂Cl₂ and the organic phases combined, dried over MgSO₄, filtered andevaporated. Purification using preparative HPLC gave Compound 96 as anoff-white solid (28 mg).

LCMS (10 cm_ESCI_Formic_MeCN) Rt 4.32 min; m/z 365 [M+H] 99.30% purity.

Example 904-chloro-3,5-diphenyl-1-(tetrahydrofuran-3-yl)-1H-pyrazolo[3,4-c]pyridazine(Compound 97)

Compound 97 was synthesised from4-chloro-3,5-diphenyl-1H-pyrazolo[3,4-c]pyridazine (50 mg, 0.16 mmol)following a similar procedure outlined in Example 86, using3-hydroxytetrahydrofuran (28 mg, 0.32 mmol) instead of oxetan-3-ol.Compound 97 was purified using preparative HPLC followed bychromatography (silica gel, gradient CH₂Cl₂), yielding Compound 97 as anoff-white solid (21 mg).

LCMS (10 cm_ESCI_Formic_MeCN) Rt 4.64 min; m/z 377 [M+H] 99.42% purity.

Example 91 Liquid Chromatography-Mass Spectrometry (LC-MS)

LC-MS Conditions for the Pyrazolopyridizines Compounds of the Invention.

(a) Standard Acidic LC-MS Conditions: (10 cm_ESI_Formic or 10cm_ESCI_Formic)

A Phenomenex Luna 5 μm C18 (2), 100×4.6 mm (plus guard cartridge) columnusing an acetonitrile (Far UV grade) with 0.1% (V/V) formic acid: water(high purity via PureLab Option unit) with 0.1% formic acid gradient wasused. The flow rate was 2 mL/min. UV detection was done using a Watersdiode array detector (start Range 210 nm, end range 400 nm, rangeinterval 4 nm). Mass detection was performed via a single quadrapoleLC-MS instrument. Ionisation is either ESI or APCI dependent on compoundtypes. The gradient used ran from 95% of aqueous solvent at time 0.00min to 5% of aqueous solvent at 3.50 min. This percentage was then heldfor a further 2 min.

(b) Standard Basic LC-MS Conditions: (10 cm_ESCI_Bicarb or 10cm_ESI_Bicarb):

A Waters Xterra MS 5 μm C18, 100×4.6 mm (plus guard cartridge) using anacetonitrile (Far UV grade): water (high purity via PureLab Option unit)with 10 mM ammonium bicarbonate (ammonium hydrogen carbonate) gradientwas used. The flow rate was 2 mL/min. UV detection was performed using aWaters diode array detector (start Range 210 nm, end range 400 nm, rangeinterval 4 nm). Mass detection was performed via a single quadrapoleLC-MS instrument. Ionisation is either ESI or APCI dependent on compoundtypes. The gradient used ran from 95% of aqueous solvent at time 0.00min to 5% of aqueous solvent at 4.0 min. This percentage was then heldfor a further 1.5 min.

(c) Standard Acidic HPLC Conditions: (15 cm_Formic_ASCENTIS_HPLC)

A Supelco Ascentis® Express C18 or Hichrom Halo C18, 2.7 μm C18, 150×4.6mm column using an acetonitrile (Far UV grade) with 0.1% (V/V) formicacid: water (high purity via PureLab Option unit) with 0.1% formic acidgradient was used. The flow rate was 1 mL/min. UV detection was doneusing an Agilent diode array detector (300 nm, band width 200 nm; ref450 nm, band width 100 nm). The gradient used ran from 96% of aqueoussolvent at time 0.00 min to 0% of aqueous solvent at 9.00 min. Thispercentage was then held for a further 4.5 min.

(d) Standard Basic HPLC Conditions: (15 cm_Bicarb_GeminiNX_HPLC)

A Phenomenex Gemini NX, 3 μm C18, 150×4.6 mm column using anacetonitrile (Far UV grade): water (high purity via PureLab Option unit)with 10 mM ammonium bicarbonate gradient was used. The flow rate was 1mL/min. UV detection was done using an Agilent diode array detector (300nm, band width 200 nm; ref 450 nm, band width 100 nm). The gradient usedran from 95.5% of aqueous solvent at time 0.00 min to 0% of aqueoussolvent at 9.00 min. This percentage was then held for a further 4.5min.

(e) Standard Acidic HPLC Conditions: (10 cm_Formic_ACE 3 C18AR_HPLC_CH3CN)

A Hichrom ACE 3 C18-AR mixed mode 100×4.6 mm column using anacetonitrile (Far UV grade) with 0.1% (V/V) formic acid: water (highpurity via PureLab Option unit) with 0.1% formic acid gradient was used.The flow rate was 1 mL/min. UV detection was done using an Agilent diodearray detector (300 nm, band width 200 nm; ref 450 nm, band width 100nm). The gradient used ran from 98% of aqueous solvent from time 0.00min to 3.00 min, to 100% of aqueous solvent at 12.00 min. Thispercentage was then held for a further 2.4 min.

Example 92 Assay Method Showing Activity of Compounds of the Inventionthat Restore Expression of N48K Clarin-1

Clarin-1 is the protein encoded by the gene mutated in Usher IIISyndrome (Adato et al., 2002). The most prevalent mutation in Clarin-1in North America is N48K, which is reported to cause loss ofglycosylation and a trafficking defect (Tian et al., 2009). As aconsequence, the N48K protein does not reach the plasma membrane and isdegraded by the proteasome. Thus it is believed that restoring thetrafficking of N48K Clarin-1 to the cell surface would provide an avenueof intervention for Usher III Sydrome.

A useful cellular model to demonstrate the utility of compounds of theinvention that restore expression of N48K Clarin-1 is theHEK293-Clarin-1 N48K-HA D9 cell line (Tian et al., 2009). In a typicalexperiment, these cells are seeded on collagen-coated 96-well plates ata cell density of 20,000 cells per well in Dulbecco's Modified EagleMedium (DMEM) contain 10% fetal bovine serum in a humidified incubatorat 37° C., 5% CO₂. After an overnight incubation, compounds are addedfor a 24 hr incubation in DMEM medium contain 10% fetal bovine serum ina humidified incubator at 37° C., 5% CO₂. As a negative control, DMSO isused at 0.25% final concentrations. Compounds are typically tested intriplicate fashion. After the 24 hr incubation with compounds, the cellsare fixed by the addition of 10% buffered formalin to the wells toachieve a final concentration of 4% formalin. After a 20 min fixation atroom temperature, wells are washed three times with phosphate-bufferedsaline (PBS) containing Triton X-100 (0.02 phosphate, 150 mM NaCl, 0.1%Triton X-100).

The HA-tagged N48K Clarin-1 is detected with an antibody against the HAtag (HA.11 Clone 16B12 Monoclonal antibody, Covance #MMS-101P) at adilution of 1:1000 in PBS containing Triton X-100. After a 90 minincubation, wells are washed three times with PBS containing TritonX-100, and a secondary antibody (Goat anti-mouse IgG-Cy3 (1.5 mg/ml),Jackson IR Europe #115165003) is added to the wells at a dilution of1:250 in PBS containing Triton X-100 for 45 min. Wells are subsequentlywashed three times with PBS containing Triton X-100, and a finalstaining for nuclei is performed by the addition of DAPI(4′,6-diamidino-2-phenylindole) at a dilution of 1:10,000. The imagingof the stained cells is performed for instance on an InCell 1000 HighContent Imager (GE Healthcare), reading out the Cy3 channel for N48KClarin-1 and the DAPI channel for nuclei. The images are analyzed andquantitated using a specific algorithm. This algorithm measures theHA-Clarin-1 staining for each cell based on the additional nuclearsegmentation of the DAPI signal (FIG. 1). This algorithm is preferred asit measures the intensity per cell, and thus it is less sensitive forvariation in cell number. Per well, approximately 2,000 cells aremeasured to achieve an average density per cell measurement.

Example 93 IC₅₀ Data for Illustrative Compounds of the Invention

IC₅₀ values for activity of illustrative Pyrazolopyrimidine compounds ofthe invention were obtained according to the assay method of Example 92.IC₅₀ values for Compounds 1-35, 37-39, 42, 44, 45, and 47-97, are lessthan or equal to 10 micromolar. IC₅₀ values for compounds 1-9, 10, 1113-34, 37-39, 44, 45, 47-97 are all less than or equal to 5 micromolar.IC₅₀ values for compounds 1, 2, 4-9, 13, 14, 17, 19, 28, 32, 38, 39, 45,48-61, 63-74, 76, 77, 79, 81, 83, 85-97 are all less than or equal to 1micromolar.

Example 94 Assay Method for Identifying One or More Proteins that Bindthe Pyrazolopyrimidine Moiety of Compound 44

Compound 44, which includes a pyrazolopyrimidine moiety and biotingroup, is useful as a probe for identifying proteins that bind to itspyrazolopyrimidine moiety. The proteins are identified as follows:HEK293-Clarin-1 N48K-HA D9 cells are seeded on collagen-coated 96-wellplates at a cell density of 20,000 cells per well in Dulbecco's ModifiedEagle Medium (DMEM) containing 10% fetal bovine serum in a humidifiedincubator at 37° C., 5% CO₂. After an overnight incubation, Compound 44is added to each well at a concentration of less than or equal to 10micromolar for a 24 hr incubation. The incubation is optionallyperformed in triplicate. After the 24 hr incubation with Compound 44,the cells are washed three times with phosphate-buffered saline (PBS)and converted to a cell lysate. The protein bound to Compound 44 is thenseparated and identified using any suitable method known in the art. Forexample, the cell lysate is applied to streptavidin-coated beads, andthe beads are washed with a buffer containing a detergent, such assodium dodecylsulfate (SDS), to remove any non-specific binding protein.The bound protein is then eluted off the beads with, for example, using8M guanidine•HCl, pH 1.5, by boiling the beads in SDS-PAGE(-polyacrylamide gel electrophoresis) buffer, or by a tryptic digest ofthe beads. The mass of the released peptides can then be measured with amass spectrometer such as Matrix Assisted Laser DesorptionIonization-Time-of-flight (MALDI-TOF) or Electrosprayionization-Time-of-flight (ESI-TOF). These masses are then compared tothose of a database containing known protein sequences in order toidentify the proteins. The peptide fragments which are bound to Compound44 are also identified, since the molecular weight of the peptidefragment increases by the molecular weight of Compound 44.

Each reference disclosed in this application is incorporated byreference herein in its entirety.

What is claimed is:
 1. A method for treating a retinal degenerativedisease, comprising administering to a subject in need thereof aneffective amount of a compound having the structure:

or a pharmaceutically acceptable salt thereof.
 2. A method for treatinghearing loss associated with Usher syndrome, comprising administering toa subject in need thereof an effective amount of a compound having thestructure:

or a pharmaceutically acceptable salt thereof.